CN116583275A - MDM2 inhibitors for the treatment or prevention of hematological tumor recurrence following hematopoietic cell transplantation - Google Patents

Abstract

The present invention relates to a mouse double minute 2 (MDM 2) inhibitor for use in the treatment and/or prevention of hematological tumor recurrence following Hematopoietic Cell Transplantation (HCT) in a patient. In embodiments, the hematological tumor is leukemia, preferably Acute Myelogenous Leukemia (AML). Preferably, the patient receives allogeneic T cell (allogeneic T cell) transplantation with and/or after HCT, e.g., at the time point of MDM2 administration. Furthermore, the present invention relates to a pharmaceutical composition comprising an MDM2 inhibitor and an exporter 1 (XPO-1) inhibitor for use in the treatment and/or prevention of a relapse of a hematological tumor following Hematopoietic Cell Transplantation (HCT) in a patient according to any of the preceding claims.

Description

MDM2 inhibitors for the treatment or prevention of hematological tumor recurrence following hematopoietic cell transplantation

Description

The present invention relates to a mouse double minute 2 (MDM 2) inhibitor for use in the treatment and/or prevention of hematological tumor recurrence following Hematopoietic Cell Transplantation (HCT) in a patient. In embodiments, the hematological tumor is leukemia, preferably Acute Myelogenous Leukemia (AML). Preferably, the patient receives allogeneic T cell (allogeneic T cell) transplantation with and/or after HCT, e.g., at the time point of MDM2 administration. Furthermore, the present invention relates to a pharmaceutical composition comprising an MDM2 inhibitor and an exporter 1 (XPO-1) inhibitor for use in the treatment and/or prevention of a relapse of a hematological tumor following Hematopoietic Cell Transplantation (HCT) in a patient according to any of the preceding claims.

Background

Acute Myelogenous Leukemia (AML) recurrence is the leading cause of death after allogeneic hematopoietic cell transplantation (allo-HCT) at day 100 post-transplantation (1). Major mechanisms that promote relapse include down-regulation of MHC class II (MHC-II) (2, 3), deletion of mismatched HLA4, up-regulation of immune checkpoint ligands (3), and reduction of IL-15 production (5), and reduction of leukemia-derived lactate release (6), among others (reviewed in 7). Down-regulation of pro-apoptotic genes, including TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2, has been shown to be associated with therapeutic resistance and recurrence in AML (8). These data indicate that methods of increasing MHC-II or TRAIL-R1/2 expression can successfully treat AML recurrence following allogeneic HCT.

Current pharmacological approaches for AML recurrence include immune checkpoint inhibitors, demethylating agents, bcl-2 inhibitors, and the like, among other FLT3 kinase inhibitors (reviewed in 9). Mouse double microsomal-2 (MDM 2) inhibitors (10, 11) can induce p 53-dependent apoptosis in AML, however, their role in post-allogeneic HCT settings has not been evaluated so far.

In light of the prior art, there remains a great need in the art to provide additional and/or improved methods for treating leukemia or lymphoma recurrence, particularly AML recurrence following HCT. In particular, such treatment may include compounds that increase MHC-II or TRAIL-R1/2 expression in leukemia cells. However, these compounds have not been available so far, and there is still a need to provide such compounds.

Disclosure of Invention

According to the prior art, the technical problem underlying the present invention is to provide alternative and/or improved means (means) for the treatment of leukemia or lymphoma recurrence, in particular AML recurrence after HCT. Such means should include compounds, molecules and/or compositions suitable for mediating up-regulation or maintenance of expression of MHC-II or TRAIL-R1/2 in leukemia cells.

This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

Accordingly, the present invention relates to a mouse double minute 2 (MDM 2) inhibitor for use in the treatment and/or prevention of hematological tumor recurrence following Hematopoietic Cell Transplantation (HCT) in a patient. The MDM2 inhibitor may be administered before and/or simultaneously with and/or after (preferably after) the administration of HCT.

The present invention is based on the entirely surprising discovery that recurrence of cancer cells in patients with hematological tumors after HCT can be specifically treated or prevented by administration of MDM2 inhibitors. The present invention also unexpectedly found that inhibition of MDM2 results in upregulation of MHC-I and MHC-II molecules, as well as upregulation of TRAIL-receptors in cancer cells, such as leukemia cells or AML cells. This results in a substantial enhancement of the recognition of patient cancer cells by allogeneic T cells that have been introduced into the patient by HCT (hematopoietic cell transplantation) and/or by transplantation of allogeneic T cells alone (allogeneic donor lymphocyte infusion; DLI). In other words, exposure to the MDM2 inhibitor renders the patient's cancer cells immunologically "visible" or strongly enhances the immunological "visibility" so that the transplanted allogeneic T cells can now recognize and attack the cancer cells.

The MDM2 protein acts as an inhibitor of ubiquitin ligases and p53 transcriptional activation, with ubiquitin ligases recognizing the N-terminal transactivation domain of p 53. MDM2 overexpression and oncogenic Ras synergistically promote transformation of primary rodent fibroblasts, and MDM2 inhibition can increase p53 activity (11). MDM2 acts by reducing p53 protein levels, which promotes the accumulation of new-born mutations (de novo mutations) in tumor cells, thereby enhancing their malignant potential. In addition to its anti-oncogenic effects, p53 can also increase the expression of certain immune-related genes. In the context of the present invention, it has surprisingly been found that in cancer cells of hematological tumors, in particular in AML cells, a similar mechanism is operable, i.e. up-regulation of HLA-class II molecules and TRAIL-receptors, rendering them more sensitive to alloreactive donor T cell responses after allogeneic HCT.

It was completely unexpected that MDM2 inhibition resulted in the expression of TRAIL-R1/2 in leukemia and lymphoma cells (e.g., primary human AML cells and AML cell lines). After TRAIL ligation, TRAIL death receptors assemble within their intracellular death domain a death-inducing signaling complex (DISC) consisting of FAS-related protein with death domain (FADD) and procaspase 8/10 (17). TRAIL-R activation was shown to have anti-tumor activity (18).

Furthermore, it was found herein that MDM2 inhibition also increases MHC-II expression on primary leukemia and lymphoma cells, particularly human AML cells, which may provide pharmacological intervention to reverse the MHC-II reduction observed in AML recurrence following allogeneic HCT (2, 3).

In embodiments, the hematological neoplasm is selected from the group consisting of leukemia, lymphoma, and myelodysplastic syndrome. In embodiments, the hematological tumor is leukemia, preferably Acute Myelogenous Leukemia (AML).

In embodiments, the hematological tumor comprises one or more mutations, such as oncogenic mutations, that induce MDM2 and/or MDM4 expression in the tumor cells. Surprisingly, certain mutations induce MDM2 and/or MDM4, which makes these tumor cancer cells particularly sensitive to treatment with MDM2 inhibitors. In a preferred embodiment, the hematological tumor comprising one or more MDM2 and/or MDM4 induced mutations is AML. The MDM2 and/or MDM4 induced mutation may be, for example, a point mutation or a fusion gene, which may be formed by chromosomal translocation.

MDM2 and/or MDM4 induced mutations may be selected from, without limitation, cKit-D816V, FIP 1L-PDGFR-alpha, FLT3-ITD and JAK2-V617F. Additional MDM2 and/or MDM4 induced mutations can be identified, for example, by using the techniques described herein.

cKit-D816V is an activating mutation at codon 816 of the Kit gene, which is involved in malignant cell growth, in particular in Acute Myelogenous Leukemia (AML), in systemic mastocytosis and germ cell tumors, characterized in that aspartic acid is replaced by valine (D816V), and which makes the receptor independent of activating and signalling ligands.

FIP1L1-PDGFR alpha fusion genes have been detected in hematological malignancies, particularly eosinophils, neutrophils, mast cells, monocytes, T lymphocytes and B lymphocytes involved in AML. FIP1L 1-PDGFR-a fusion proteins retain PDGFR-a-related tyrosine kinase activity, but unlike PDGFR-a their tyrosine kinases are constitutive, i.e. continuously active: fusion proteins lack the intact membrane-proximal domain of PDGFR-a, which normally blocks tyrosine kinase activity, unless PDGFR-a binds to its activating ligand, the platelet-derived growth factor. FIP1L 1-PDGFR-alpha fusion proteins are also resistant to the normal degradation pathway of PDGFR-alpha, i.e. proteasome-dependent ubiquitination. Thus, they are highly stable, long-lived, unregulated, and continue to express the stimulatory effects of their PDGFRA tyrosine kinase components.

Hematopoietic tumor recurrence (e.g., AML recurrence) following treatment of HCT with an MDM2 inhibitor, preferably in combination with allogeneic T cell transplantation, is particularly effective in patients with tumors harboring MDM2 and/or MDM4 induced mutations. Thus, in a preferred embodiment, the patient is known to have a hematopoietic tumor carrying these mutations (e.g., FLT3-ITD, JAK2-V617F, cKit-D816V, or FIP 1L-PDGFR-alpha).

In embodiments, the HCT is allogeneic HCT. Preferably, the hematopoietic cell transplant is allogeneic (and most preferably not T cell depleted) in that, due to differences in HLA molecules, allogeneic T cells contained in the transplant can produce a graft versus leukemia or graft anticancer cellular response to cancer cells that recur after HCT. Thus, administration of MDM2 inhibitors may result in a stronger anti-cancer effect of transplanted T cells against cancer cells and may prevent recurrence of cancer after HCT or may result in control or eradication of cancer cells after recurrence occurs.

In embodiments, HCT comprises T cells.

In embodiments, the MDM2 inhibitor is administered to the patient after HCT and before recurrence occurs. In the context of the present invention, MDM2 inhibitors may be administered to a patient at different time points. For example, the inhibitor may be administered at the time point of HCT (the time point of hematopoietic cell transplantation), for example, on the same day. In embodiments, it may be useful to have an inhibitor administered prior to HCT (e.g., 1, 2, 3, 4, 5, 6, or 7 days prior to HCT) so that the remaining cancer cells are immediately visible to T cells contained in the hematopoietic cell transplant. MDM2 inhibitors may also be found in HCT

18. 19, 20 days or more). In some embodiments, the MDM2 inhibitor is administered before, and after the administration of HCT. Preferably, the MDM2 inhibitor is administered (only) after HCT administration.

MDM2 inhibitor administration occurs multiple times and even is repeated periodically, e.g., once a day, once every other day, once every 4 days, once a week, once a month, (repeated) days 1-5 of a 28-day schedule, or (repeated) days 1-7 of a 28-day schedule.

MDM2 inhibitor administration may routinely occur in patients with hematological tumors who have received and/or are receiving and/or will receive HCT as a prophylactic measure, for example, to enhance graft versus cancer effects and prevent the occurrence of cancer recurrence in the patient.

In embodiments, the inhibitor is administered to the leukemia patient after recurrence occurs after HCT. MDM2 inhibitor administration may be a post-recurrence treatment for hematological tumor patients following HCT, possibly in combination with further allogeneic T cell transplantation, preferably Donor Lymphocyte Infusion (DLI) without hematopoietic stem cells.

In one embodiment, the MDM2 inhibitor is administered after HCT, and a) before allogeneic T cell transplantation, and/or b) on the same day as allogeneic T cell transplantation, and/or c) after allogeneic T cell transplantation.

In this context, it should be understood that the combined administration of an MDM2 inhibitor and allogeneic T cell transplantation may involve the coordinated administration of the inhibitor and the cells. The two products need not be applied in a single composition, but may also be applied as separate compositions at different points in time. For example, a patient may first receive an MDM2 inhibitor to induce up-regulation of, for example, TRAIL-R1, TRAIL-R2, human Leukocyte Antigen (HLA) class I molecules, and HLA class II molecules, and then receive T cell transplantation, for example, after the same day, or after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. However, both products may also be administered at about the same time, meaning that they are administered within about 8 hours, or the MDM2 inhibitor may be administered after T cell transplantation administration. Herein, one or both products (MDM 2 inhibitor or T cell graft) may be administered to the patient multiple times in a synergistic manner.

It is to be understood that in the context of the present invention, the synergistic administration of MDM2 with other products such as HCT, allogeneic T cell transplants and/or XPO-1 inhibitors involves the administration of MDM2 inhibitors and other products to enhance the therapeutic or prophylactic effect of the inhibitors. Those skilled in the art will be able to coordinate the respective administration of the inhibitor and the other compounds/products according to the appropriate administration regimen for the patient receiving the MDM2 inhibitor. In addition, it is likely that leukemias with certain mutations that induce MDM2 expression will respond particularly well because, for example, cKIT-D816V and FIP 1L-PDGFR-alpha are observed to induce MDM2 and MDM4. From this, it can be seen that the allogeneic T cell/MDM 2 inhibitor combination after allogeneic HCT (bone marrow transplantation) was highly effective in mice carrying FIP1L-PDGFR- α mutant and cKIT-D816V mutant AML.

In embodiments, the treatment of the invention further comprises administering allogeneic T cell transplantation with and/or after the HCT. In embodiments, allogeneic T cell transplantation is donor lymphocyte infusion that includes lymphocytes but does not include hematopoietic stem cells. In embodiments, the donor for allogeneic T cell transplantation is also a donor for HCT.

In the context of the present invention, the MDM2 inhibitor is preferably selected from the group comprising RG7112 (RO 5045337), idaneanlin (RG 7388), AMG-232 (KRT-232), APG-115, BI-907828, CGM097, siremalin (HDM-201) and Mi Lade maytansine (milademan, DS-3032 b) and pharmaceutically acceptable salts thereof. In one embodiment, the MDM2 inhibitor is cisco-crystal (e.g., succinic acid co-crystal or succinate salt) or a pharmaceutically acceptable salt or co-crystal thereof.

Various MDM2 inhibitors are known in the art and a number of established assays for identifying MDM2 inhibitors have been described and are being investigated for the treatment of various disorders (Marina Konopleva et al, leukemia.2020Jul 10.Doi:10.1038/s 41375-020-0949-z). However, the use of MDM2 inhibitors for the specific treatment or prevention of cancer recurrence in hematological tumor patients following HCT has not been described or suggested in the art. The advantages of this treatment have not been described so far and are based on the completely surprising finding that up-regulation of cancer cells of a hematological tumor, such as leukemia cells, enhances the recognition of cancer cells by allogeneic T cells.

In embodiments, administration of the MDM2 inhibitor results in upregulation of one or more of TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1), TRAIL-R2, human Leukocyte Antigen (HLA) class I molecules, and HLA class II molecules. Thus, in embodiments, inhibition of MDM2 results in upregulation of one or more of TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1), TRAIL-R2, human Leukocyte Antigen (HLA) class I molecules, and HLA class II molecules. In embodiments, up-regulation of one or more of TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1), TRAIL-R2, human Leukocyte Antigen (HLA) class I molecules and HLA class II molecules, in particular up-regulation of TRAIL-R1 and/or TRAIL-R2, is p53 dependent.

In embodiments, administration of the MDM2 inhibitor increases the cytotoxicity of cd8+ allogeneic T cells against cancer cells, wherein preferably the cytotoxicity of cd8+ allogeneic T cells is dependent at least in part on the interaction of TRAIL-R of cancer cells and TRAIL-ligand (TRAIL-L) of cd8+ allogeneic T cells.

In embodiments, administration of the MDM2 inhibitor increases a Graft Versus Leukemia (GVL) or graft versus lymphoma response, wherein preferably the graft versus leukemia response or graft versus lymphoma response is mediated by cd8+ allogeneic T cells.

In embodiments, administration of the MDM2 inhibitor increases expression of one or more of perforin, CD107a, IFN- γ, TNF, and CD69 by cd8+ allogeneic T cells. Thus, according to one aspect of the invention, provided herein is a method of increasing expression of one or more of perforin, CD107a, IFN- γ, TNF and CD69 by cd8+ allogeneic T cells, the method comprising administering an MDM2 inhibitor (e.g., HDM201 or a pharmaceutically acceptable salt thereof) in combination with HCT (e.g., allogeneic HCT, e.g., comprising T cells).

In embodiments, administration of the MDM2 inhibitor induces a longevity profile of T cells, particularly cd8+ T cells such as cd8+ allogeneic T cells (as described in (13)). For example, in embodiments, transplanted CD8+ T cells display high expression of Bcl-2 and/or IL-7R (CD 127) in the context of MDM2 inhibition. Furthermore, in embodiments, administration of the MDM2 inhibitor induces cd8+ T cells (as defined, for example, (12)) with a high antigen recall response, such as cd8+ T cells lacking CD 27. In embodiments, MDM2 inhibitor treatment induces a decrease in cd8+cd27+tim3+ donor T cells.

It is another totally unexpected finding of the present invention that administration of MDM2 inhibitors not only results in upregulation of receptors and surface molecules on cancer cells as described herein, but can also induce an advantageous phenotype in allogeneic T cells of the patient, resulting in a stronger cytotoxic effect of T cells on cancer cells. Roughly speaking, MDM2 inhibitors can induce a more cytotoxic phenotype in cd8+ allogeneic T cells, making them more "aggressive" to recurrent cancer cells. Thus, according to one aspect of the invention there is thus provided a method of inducing a more potent cytotoxic phenotype in cd8+ allogeneic T cells, the method comprising administering an MDM2 inhibitor (e.g. HDM201 or a pharmaceutically acceptable salt thereof) in combination with HCT (e.g. allogeneic HCT, e.g. comprising T cells).

In embodiments, administration of an MDM2 inhibitor according to the invention to a subject enhances glycolytic activity of T cells in vivo during a graft versus leukemia response. Thus, in embodiments, MDM2 inhibition results in an increase in glycolytic activity of T cells in a subject. Thus, according to one aspect of the invention there is thus provided a method of enhancing glycolytic activity in cd8+ allogeneic T cells, the method comprising administering an MDM2 inhibitor (e.g. HDM201 or a pharmaceutically acceptable salt thereof) in combination with HCT (e.g. allogeneic HCT, e.g. comprising T cells).

As shown herein, MDM2 inhibition results in increased glycolytic activity in T cells (including cytotoxic T cells), which indicates stronger T cell activation and increased GVL activity. In embodiments, the MDM2 inhibitor treatment increases activation of T cells and/or increases GVL activity of T cells in the subject. The T cells may be endogenous or administered T cells, preferably cd8+ allogeneic T cells. As shown in the examples below, MDM inhibition in a subject induces an increase in glycolytic activity of T cells in the subject.

It is entirely unexpected that administration of MDM2 inhibitors in the context of the present invention induces a T cell phenotype with enhanced/increased glycolytic activity, further enhancing the cytotoxic activity of cd8+ allogeneic T cells.

In embodiments, the patient may additionally receive an export protein 1 (XPO-1) inhibitor. Thus, in an embodiment, the invention relates to an MDM2 inhibitor for use according to the invention, wherein said treatment further comprises administration of an export protein 1 (XPO-1) inhibitor.

As shown in the examples below, MDM2 inhibition in AML cells results in increased TRAIL-R1/2 expression and enhanced GVL for AML cells, which is a great advantage in patients who relapse after treatment of HCT or in the case of preventing relapse after HCT. The molecule XPO-1 mediates the export of p53 from the nucleus and it has surprisingly been found that in certain cancer cells XPO-1 reduces p53 induced TRAIL-R1/2/MHC-II production following MDM2 inhibition. It is therefore advantageous in the context of the present invention to additionally inhibit XPO-1 to maximize the effect of MDM2 inhibition. The MDM2 inhibitor and XPO-1 inhibitor may be administered in a synergistic manner as described above for the combined administration of the MDM2 inhibitor and the hematopoietic cell transplant or allogeneic T cell transplant. Administration of the two inhibitors may be performed alone or in the form of a pharmaceutical product or composition comprising the two inhibitors.

Accordingly, the present invention also relates to a pharmaceutical composition comprising an MDM2 inhibitor and an exporter 1 (XPO-1) inhibitor for use in the treatment and/or prevention of a relapse of a hematological tumor following Hematopoietic Cell Transplantation (HCT) in a patient according to any of the preceding claims. Such pharmaceutical compositions may be used in the context of all embodiments described herein.

Furthermore, according to one aspect of the present invention there is thus provided an XPO-1 inhibitor for use in the treatment and/or prophylaxis of hematological tumors in a patient, wherein the treatment further comprises administration of a hematopoietic cell transplant (e.g. allogeneic, e.g. comprising T cells) and an MDM2 inhibitor.

Detailed Description

All cited patent and non-patent documents are incorporated herein by reference in their entirety.

Accordingly, the present invention relates to a mouse double minute 2 (MDM 2) inhibitor for use in the treatment and/or prevention of hematological tumor recurrence following Hematopoietic Cell Transplantation (HCT) in a patient.

As used herein, "prevention" of hematological tumor recurrence should be understood to refer to any method, process, or action that is intended to ensure that hematological tumor recurrence does not occur. Prevention relates to prophylactic treatment aimed at avoiding recurrent conditions. "prophylactic" treatment is treatment administered to a subject who exhibits no or only early signs of disease, with the aim of reducing the risk of developing pathology, in this case occurrence of recurrence after HCT.

The term "treatment" refers to a therapeutic intervention that ameliorates signs or symptoms of a disease or pathological condition (here, recurrence of a hematological tumor following HCT) after its onset. As used herein, the term "ameliorating" with respect to a disease or pathological condition refers to any observable beneficial effect of a treatment. The beneficial effect may be demonstrated, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in the severity of some or all of the clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters specific to a particular disease that are well known in the art.

As used herein, the terms "subject" and "patient" include human and veterinary subjects, particularly mammals and other organisms. The term "receptor" relates to a patient or subject receiving HCT and an MDM2 inhibitor of the invention.

It should be understood that the term "tumor" relates to a new abnormal growth of tissue. Malignant tumors exhibit a greater degree of hypoplasia than benign tumors, and have invasive and metastatic properties. As used herein, the term "hematological tumor" relates to a tumor located in blood and blood-forming tissue (bone marrow and lymphoid tissue). The most common forms are various types of leukemias, lymphomas and myelodysplastic syndromes, particularly progressive life-threatening myelodysplastic syndromes.

The term hematological neoplasm includes neoplasms and cancers of the hematopoietic and lymphoid tissues associated with neoplasms and cancers affecting the blood, bone marrow, lymph and lymphoid system. Because hematopoietic and lymphoid tissues are tightly linked by the circulatory and immune systems, diseases affecting one of them often affect the other as well, making myeloproliferation and lymphoproliferation (and thus leukemia and lymphoma) closely related and often overlapping problems.

Hematological malignancies as subject of the present invention are malignant ("cancer") and they are usually treated by specialists in hematology and/or oncology, as the affiliated profession of medical, surgical and radiological oncologists are also associated with these disorders. Hematological malignancies may be derived from either of two major blood cell lines, bone marrow and lymphoid cell lines. Bone marrow cell lines generally produce granulocytes, erythrocytes, platelets, macrophages and mast cells; lymphoid cell lines produced B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias and myelomas are from lymphoid systems, while acute and chronic myelogenous leukemias, myelodysplastic syndromes and myeloproliferative disorders are from bone marrow.

In the context of the present invention, leukemia includes, but is not limited to, acute non-lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, basophilic leukemia, lymphoblastic leukemia, bovine leukemia, chronic myelogenous leukemia, cutaneous leukemia, embryonal leukemia, eosinophilic leukemia, grosven leukemia, hairy cell leukemia, hematogenic leukemia, histiogenic leukemia, stem cell leukemia, acute monocytic leukemia leukopenia, lymphoblastic leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryoblastic leukemia, microsomal leukemia, monocytic leukemia myeloblastic leukemia, myelogenous leukemia, myeloblastic leukemia, myelomonocytic leukemia, naegeli leukemia, plasma cell leukemia, promyelocytic leukemia, rieder cell leukemia, schiling leukemia, stem cell leukemia, sub-leukemia, and undifferentiated cell leukemia.

According to the invention, lymphomas include hodgkin and non-hodgkin lymphomas (B-cell and T-cell lymphomas), including but not limited to diffuse large B-cell lymphomas (DLBCL), primary mediastinal B-cell lymphomas, follicular lymphomas, chronic lymphocytic leukemias, small lymphocytic lymphomas, mantle cell lymphomas, marginal zone B-cell lymphomas, junction outer border zone B-cell lymphomas (also known as mucosa-associated lymphoid tissue (MALT) lymphomas), junction marginal zone B-cell lymphomas and spleen marginal zone B-cell lymphomas, burkitt lymphomas, lymphoplasmacytic lymphomas (Waldenstrom's macroglobulinemia), hairy cell leukemia primary Central Nervous System (CNS) lymphomas, precursor T lymphoblastic lymphomas/leukemias, peripheral T-cell lymphomas, cutaneous T-cell lymphomas (mycosis, sezary syndrome, etc.), adult T-cell leukemias/lymphomas, including stasis, chronic, acute and lymphomas subtypes, angioimmunoblastic T-cell lymphomas, eater-cell lymphomas, peripheral T-cell lymphomas, and peri-cell lymphomas (eatic lymphomas), and peri-cell lymphomas (peri-cell lymphomas).

Myelodysplastic syndrome (MDS) is a group of cancers in which immature blood cells in the bone marrow are immature and therefore do not become healthy blood cells. Symptoms may include sensory fatigue, shortness of breath, susceptibility to bleeding, or frequent infection. Some types may develop into acute myelogenous leukemia.

Acute Myelogenous Leukemia (AML) is a cancer of the myeloid lineage of blood cells, characterized by the rapid growth of abnormal cells that accumulate in the bone marrow and blood and interfere with normal blood cell production. Symptoms may include sensory fatigue, shortness of breath, susceptibility to bruising and bleeding, and increased risk of infection. Sometimes, diffusion may occur in the brain, skin or gums. AML progresses rapidly as acute leukemia and is usually fatal within weeks or months if left untreated. AML is usually initially treated with chemotherapy with the aim of inducing remission. One can then continue to receive additional chemotherapy, radiation therapy, or stem cell transplantation. The presence of specific genetic mutations within cancer cells can guide treatment and determine how long the human may survive.

Invasive forms of hematological tumors and hematological malignancies require treatment with chemotherapy, radiation therapy, immunotherapy, and bone marrow transplantation, a form of Hematopoietic Cell Transplantation (HCT).

Hematopoietic Cell Transplantation (HCT), also known as Hematopoietic Stem Cell Transplantation (HSCT), is the transplantation of pluripotent hematopoietic stem cells, typically derived from bone marrow, peripheral blood, or umbilical cord blood. HCT can be autologous (using the patient's own stem cells), allogeneic (stem cells from the donor) or syngeneic (from syngeneic twins). HCT is used in patients with certain cancers of the blood or bone marrow or lymphatic system such as multiple myeloma or leukemia. In these cases, the immune system of the recipient is typically destroyed completely (or in some cases only partially) by radiation and/or chemotherapy or other methods known in the art prior to implantation of the hematopoietic stem cell graft (bone marrow ablation or partial bone marrow ablation). Infection and graft versus host disease are major complications of allogeneic HCT. HCT is a dangerous procedure with many possible complications, and is therefore almost exclusively performed on patients suffering from life threatening diseases.

In the context of the present invention, HCT is preferably allogeneic. The risk of cancer exacerbation/recurrence is reduced compared to autologous HCT. Allogeneic HCT involves a (healthy) donor and a (patient) recipient. Allogeneic HCT donors must have a tissue type (human leukocyte antigen, HLA) that matches the recipient. Matching is typically performed based on variability of three or more sites of the HLA gene, with perfect matching of these sites being preferred. Even though these critical alleles match well, the recipient requires immunosuppressive drugs to alleviate graft versus host disease. Allograft donors may be related (typically HLA-closely matched siblings) or unrelated (unrelated and found to be closely matched to HLA). Umbilical cord blood was also used as a source of stem cells for allograft. In general, allogeneic HCT appears to increase the chances of cure or long-term remission once immediate transplantation-related complications are resolved by infusion of healthy stem cells into the recipient's blood stream to remodel the healthy immune system.

Compatible donors were found by conducting additional HLA tests from the blood of potential donors. HLA genes are classified into two classes (type I and type II). In general, mismatches in type I genes (i.e., HLA-A, HLA-B or HLA-C) increase the risk of graft rejection. Mismatches in HLA type II genes (i.e., HLA-DR or HLA-DQB 1) increase the risk of graft versus host disease.

Possible sources of donor cells include, but are not limited to, bone marrow, peripheral blood stem cells, amniotic fluid, and umbilical cord blood.

Graft Versus Host Disease (GVHD) is an inflammatory disease that is characteristic of allograft transplantation and is mediated by the attack of "new" bone marrow immune cells against recipient tissue. This may occur even if the donor and recipient are HLA identical, as the immune system can still recognize other differences between their tissues. Acute graft versus host disease usually occurs 3 months after implantation and may involve the skin, intestines or liver. High doses of corticosteroids, such as prednisone, are standard treatments; however, such immunosuppressive treatments often result in fatal infections. Chronic graft versus host disease may also develop after allograft transplantation and is a major source of late treatment-related complications, although it is less responsible for death.

In embodiments of the invention, the transplanted allogeneic T cells mediate an enhanced graft anti-tumor effect (GvT) through MDM2 inhibition as described herein. GvT effects occur after allogeneic HCT. The graft may contain donor T cells (T lymphocytes) which may benefit the recipient by eliminating residual malignant cells, and in the context of the present invention, the patient may receive one or more additional allogeneic T cell transplants.

GvT may develop after recognition of tumor-specific or receptor-specific alloantigens. It can result in remission or immune control of hematological malignancies and thus can be used to prevent or treat hematological tumor recurrence following HCT. This effect is applicable to myeloma and lymphoid leukemias, lymphomas, multiple myelomas and possibly breast cancers, and may be referred to in the context of the present invention as graft versus leukemia effect or graft versus lymphoma effect or graft versus multiple myeloma effect. It is closely related to graft versus host disease (GvHD) because the underlying principles of alloimmunity are the same. Cd4+cd25+ regulatory T cells (tregs) may be used to inhibit GvHD without losing beneficial GvT effects, and one skilled in the art would be able to modulate specific embodiments of the present invention to fine tune GvT effects. GVT is most likely involved in the response to polymorphic minor histocompatibility antigens expressed more extensively on many histocyte or tumor-associated antigens that are specifically expressed on hematopoietic cells. GVT is mediated primarily by Cytotoxic T Lymphocytes (CTLs), but it can be used as a sole effector by natural killer cells (NK cells).

Graft versus leukemia (GvL) is a special type of GvT effect and is a response to host leukemia cells that may remain and/or expand after myeloablative therapy before HCT causes patient relapse. GvL requires genetic differences because this effect is dependent on alloimmune principles and is part of the graft versus host response. Whereas graft versus host disease (GvHD) has a negative impact on the host, gvL is beneficial to hematopoietic malignancy patients. After HCT, both GvL and GvHD can develop. The correlation of these two effects can be seen by comparing leukemic recurrence after HCT with the progression of GvHD. Patients who develop chronic or acute GvHD have a lower chance of leukemia recurrence. GvHD can be partially prevented when T cell depleted stem cell grafts are transplanted, but at the same time the GvL effect is also reduced, as T cells play an important role in both effects. Thus, in the context of the present invention, T cell depletion is not preferred. The potential for the GvL effect in the treatment of hematopoietic malignancies is limited by GvHD. The ability to induce GvL instead of GvH after HCT would be very beneficial for those patients. There are some strategies to suppress post-transplant GvHD or to enhance GvL, but none of them provide an ideal solution to this problem. However, the use of MDM2 inhibitors as described herein represents a new strategy capable of promoting GvL and GvT reactions.

For some forms of hematopoietic malignancies, such as Acute Myelogenous Leukemia (AML), the essential cells during HCT are NK cells that interact with KIR receptors in addition to the T cells of the donor. NK cells are within the first cell to regenerate the host's bone marrow, meaning that they play an important role in implant implantation. Their alloreactivity is required due to their role in the GvL effect. Because KIR and HLA genes are independently inherited, an ideal donor may have compatible HLA genes and KIR receptors that simultaneously induce an allogeneic response to NK cells. This will occur in most unrelated donors.

When non-depleting T cell grafts are used, cyclophosphamide is used after the graft to prevent GvHD or graft rejection. Other strategies currently in clinical use for inhibiting GvHD and enhancing GvL are, for example, optimization of transplantation conditions or post-transplantation Donor Lymphocyte Infusion (DLI). One possibility is to use cytokines. Granulocyte colony-stimulating factor (G-CSF) is used to mobilize HSCs and mediate T cell tolerance during transplantation. G-CSF can help to enhance GvL effects and inhibit GvHD by reducing the levels of LPS and TNF- α. The use of G-CSF also increases Treg levels, which also helps prevent GvHD. Other cytokines may also be used to prevent or reduce GvHD without eliminating GvL, such as KGF, IL-11, IL-18, and IL-35.

Since allogeneic HCT represents an intensive curative treatment for high-risk malignancies, its inability to prevent recurrence leaves little choice for successful salvage therapy. While many patients have high early mortality due to relapse, some respond and have sustained relief, and few have a second chance of being cured with appropriate therapy. The present invention represents a novel strategy for the treatment and prevention of recurrence after HCT, as MDM2 inhibition increases the visibility of allogeneic T cell-carrying or recurrent cancer cells. Prognosis of recurrent hematological malignancy after HCT is largely dependent on four factors: the time from SCT to relapse (relapse occurs within 6 months, prognosis is worst), disease type (chronic leukemia and some lymphomas have a second possibility of further treatment cure), disease burden and site of relapse (treatment is better if the disease is treated early), and condition of the first transplant (with better outcome for patients who have the opportunity to increase alloimmunity, specificity of anti-leukemia effect with targeting agent, or modulation intensity in the second transplant). These features are directed to improved secondary grafts, chemotherapy, targeted anti-leukemia therapy, immunotherapy or palliative care treatments. Recurrence after HCT is an important issue in oncology, and the skilled artisan is aware of current understanding of the pathological mechanisms leading to recurrence, current treatment options, and patient management in cases of recurrence after HCT, as reviewed by Barrett et al (Expert Rev Hematol.2010Aug;3 (4): 429-441.Doi:10.1586/ehm. 10.32).

The mouse double minute 2 homolog (MDM 2) is also known as E3 ubiquitin-protein ligase MDM2 and is a protein encoded by the MDM2 gene in humans. MDM2 is an important negative regulator of p53 tumor suppressor and acts as both an E3 ubiquitin ligase that recognizes the N-terminal transactivation domain (TAD) of p53 tumor suppressor and as an inhibitor of p53 transcriptional activation.

MDM2 is also required for organ development and tissue homeostasis, as nonresistant p53 activation leads to p53 overactivation-dependent cell death, known as podophyllosis (podoptosis). Podocyte apoptosis is caspase independent and therefore different from apoptosis. Mitogenic effects of MDM2 are also essential for wound healing following tissue injury, while MDM2 inhibits re-epithelialization following damage to epithelial injury. Furthermore, MDM2 has a p53 independent transcription factor-like effect in nuclear factor- κβ (nfkb) activation. Thus, MDM2 promotes tissue inflammation and MDM2 inhibition has potent anti-inflammatory effects in tissue injury. Thus, MDM2 blockade has primarily anti-inflammatory and anti-mitotic effects, which may have additive therapeutic efficacy in inflammatory and hyperproliferative disorders such as certain cancers or lymphoproliferative autoimmunity such as systemic lupus erythematosus or crescentic glomerulonephritis. The key target of Mdm2 is the p53 tumor suppressor. Mdm2 has been identified as a p 53-interacting protein that inhibits the transcriptional activity of p 53. Mdm2 achieves this inhibition by binding to and blocking the N-terminal transactivation domain of p 53. Mdm2 is a p53 responsive gene, i.e. its transcription can be activated by p 53. Thus, when p53 is stabilized, transcription of Mdm2 is also induced, resulting in higher levels of Mdm2 protein. The function of MDM2 and its role in cancer is the subject of extensive research and has been reviewed in the art, for example, by Li (Front.Pharmacol., 07May 2020,volume 11,Article 631, "Targeting Mouse Double Minute 2:Current Concepts in DNA Damage Repair and Therapeutic Approaches in Cancer") et al. The same article also reviews MDM2 inhibitors currently being used in clinical studies for the treatment of various cancers. The present invention includes the use of the inhibitors discussed in this publication for the treatment and/or prevention of post-HCT hematological tumor recurrence.

The function of MDM2 has been identified as a promising target for designing inhibitors for use as anticancer drugs. Given the shortcomings of single targeted drugs in maintaining therapeutic effects over time and the conductivity of alternative signaling pathways that activate promoting resistance, dual or multiple targeted MDM2 inhibitors have emerged. Many different MDM2 inhibitors have been successfully developed for clinical trials, so that the meaning of the term "MDM2 inhibitor" is clear to the person skilled in the art, and also a number of examples of such inhibitors known in the art can be easily identified. These include, for example, RG7112 (RO 5045337), idanealin (RG 7388), AMG-232 (KRT-232), APG-115, BI-907828, CGM097, circeide (HDM-201) and Mi Lade Meptan (DS-3032 b).

Nutlin is a series of cis-imidazoline analogs that were identified as binding to MDM2 in the p 53-binding pocket, resulting in cell cycle arrest and apoptosis in cancer cells, and growth inhibition of human tumor xenografts in nude mice. Several inhibitors such as RG7112, RG7388, RG7775, SAR405838, HDM201, APG-115, AMG-232 and MK-8242 have recently been developed to target MDM2-p53 to treat human cancers through clinical trials.

RG7112

Is a small molecule MDM2 inhibitor which enters human clinical test for the first time and is derived from the structural modification of Nutlin-3 a. RG7112 was designed to target MDM2 in the p53 binding pocket and restore p53 activity, inducing strong p21 expression and apoptosis in p53 wild-type glioblastoma (glioblastomas) cells. To date, 7 clinical studies on RG7112 have been completed (http:// www.clinicaltrials.gov/; NCT01677780, NCT01605526, NCT01143740, NCT01164033, NCT00559533, NCT00623870, NCT 01677780). The study of NP25299 (NCT 01164033) is a random crossover study of open-label of solid tumor patients. The effect of food on the pharmacokinetics of single oral dose RG7112 was evaluated. The study included two parts: the first involves an initial single dose, while the other involves four different treatment regimens with increasing doses. The results indicate that RG7112 is generally well tolerated for GI toxicity (most common AE) making it treatable with antiemetics (Patnaik et al, 2015).

A second generation Nutlin, RG7388,

to improve the efficacy and toxicity profile of early Nutlin. RG7388 induced p21 expression and efficient cell cycle arrest in MCF-7, U-2OS and SJSA-1, which confirmed activation of p 53. RG7388 is currently undergoing several clinical trials, including the only phase III clinical trial of MDM2 inhibitors (MIRROS/NCT 02545283). Results of phase I clinical trials show that RG7388 improves clinical outcome by modulating p53 activity in AML patients with high levels of MDM2 expression. MIRROS is a randomized phase III clinical trial aimed at evaluating the efficacy of RG7388 in combination with cytarabine in the treatment of relapsed and refractory Acute Myelogenous Leukemia (AML). By month 4 of 2019, the study recruited about 90% of the patient population and was still in progress. If 80% of deaths were observed in the p53-WT population of the study, an interim efficacy analysis could be obtained in 2020. MIRROS can obtain first phase III clinical trial data for MDM2 inhibitors, providing new treatment options for AML patients.

RG7775 is an inactive pegylated prodrug of AP (idaneanntin) that cleaves the pegylated tail of esterases in the blood. AP is a potent and selective inhibitor of p53-MDM2 interactions to activate the p53 pathway and is associated with cell cycle arrest and/or apoptosis. In preclinical trials, intravenous (IV) RG7775 (RO 6839921) showed anti-tumor effects in osteosarcoma and AML in immunocompromised mouse models. In phase I studies (NCT 02098967), the safety, tolerability and pharmacokinetics of RG7775 in patients with advanced malignancy were studied. The results showed that RG7775 has safety comparable to oral idanealin.

SAR405838

Is an oral selective spirooxindole small molecule derivative antagonist of MDM2 that targets MDM2-p53 interactions. In the treatment of dedifferentiated liposarcoma cells, SAR405838 is effective in stabilizing p53, activating p53 pathway, blocking cell proliferation, promoting cell cycle arrest and inducing apoptosis. SAR405838 has been used in two clinical trials in cancer patients (NCT 01636479, NCT 01985191). The study of TED12318 (NCT 01636479) is a phase I, open label, dose range, dose escalation, safety study for oral administration to adult patients with advanced solid tumors. In this trial, 74 patients received SAR405838 treatment, of which 56% showed the best response, with a 3 month no progress rate of 32%. This study showed that SAR405838 has acceptable safety in patients with advanced solid tumors. Another clinical trial with SAR405838 is a study of TCD13388 (NCT 01985191) that analyzed the safety and efficacy of SAR405838 in combination with pimasetinib (pimasertib) in cancer patients. The study was organized into 26 patients with locally advanced or metastatic solid tumors, which were demonstrated to have wild-type p53 and RAS or RAF mutations. The study was aimed at exploring the Maximum Tolerated Dose (MTD). Patient response was observed with SAR405838 at 200 or 300mg QD plus pitavastatin 60mg QD or 45mg BID. The most frequently occurring adverse events observed were diarrhea (81%), creatine phosphate kinase in blood (77%), nausea (62%) and vomiting (62%). This study shows that the safety of SAR405838 in combination with pimarictinib is consistent with the safety of both drugs.

HDM201/>

Also known as sirolimus or NVP-HDM201, is a potent and selective small molecule that inhibits the interaction between MDM2 and p53, resulting in tumor regression in preclinical models of low and high dose regimens. Compounds having similar activity and related compounds have been widely described in WO2013/111105A1 and WO2019/073435A 1. HDM201 has specific and potent killing effects on p53 wild-type cells with positive ITDs when used in combination with midotaline. HDM201 has been used in cloning experiments (NCT 02143635). NCT02143635 determines and evaluates the safe and tolerated dose of HDM201 in patients with advanced tumors with wild-type p 53. At the time of data cutoff (day 1 of month 2016), 74 patients received HDM201 (38 patients were Reg1, 36 patients were Reg2, and still receive treatment). The results show that the common grade 3/4 Adverse Events (AE) for both regimens (Reg 1 and Reg 2) were anemia (8%; 17%), neutropenia (26%; 14%) and thrombocytopenia (24%; 28%). Preliminary data indicate that hematological toxicity is delayed and dependent on regimen, and Reg1 regimen allows higher cumulative doses.

APG-115

Is a novel oral active small molecule MDM2 inhibitor. APG-115 resumes p53 expression upon binding to MDM2 and activates p 53-mediated apoptosis in tumor cells with wild-type p 53. APG-115 has been used in clinical trials to treat solid tumors (NCT 02935907), metastatic melanoma (NCT 03611868) and salivary gland carcinoma (NCT 03781986). Study NCT02935907 is a phase I study of the safety, pharmacokinetic and pharmacodynamic properties of oral APG-115 in patients with advanced solid tumors or lymphomas. Different dosage levels (including 10mg, 20mg, 50mg, 100mg, 200mg and 300 mg) were tested in this study. The results showed that the optimal dose of APG-115 was 100mg without dose limiting toxicity. In recent studies, APG-115 mediates anti-tumor immunity of the Tumor Microenvironment (TME). APG-115 activates p53 and p21 on bone marrow-derived macrophages in vitro and reduces the number of immunosuppressive M2 macrophages by down-regulating c-Myc and c-Maf. Furthermore, APG-115 showed costimulatory activity in T cells and increased expression of PD-L1 in tumor cells. This evidence suggests that a combination of APG and immunotherapy may be a new anti-tumor regimen.

AMG 232

Is a research oral selective MDM2 inhibitor that restores p53 tumor inhibition by blocking MDM2-p53 interactions. The activity of AMG232 and its effect on p53 signaling was characterized in several preclinical tumor models. AMG232 binds MDM2, strongly inducing p53 activity, leading to cell cycle arrest and inhibiting tumor cell proliferation. Several clinical trials of AMG232, such as NCT01723020, NCT02016729, NCT02110355, NCT03031730, NCT03041688, NCT03107780 and NCT03217266, have been ongoing to treat human cancers. NCT02016729 is an open label phase I study evaluating AMG232 safety, pharmacokinetics and MTD. In this study, AMG232 was administered in two regimens (group 1 and group 2). In group 1, patients were treated once daily with 60, 120, 240, 360, 480 or 960mg of AMG232 as monotherapy for 7 days every 2 weeks, or in group 2 with 60mg in combination with 2mg of trimetinib. The results show that common treatment-related AEs included nausea (58%), diarrhea (56%), vomiting (33%) and loss of appetite (25%). However, the MTD of AMG232 is not reached. Dose escalation was stopped due to its unacceptable gastrointestinal AE at higher doses.

MK-8242

Is a potent small molecule inhibitor that targets MDM2-p53 interactions. MK-8242 induces tumor regression and complete or partial response of various solid tumor types in most acute lymphoblastic leukemia xenografts. MK-8242 has been used in two phase I clinical trials (NCT 01451437 and NCT 01463696). The study of NCT01451437 is that of MK-8242 alone and in combination with cytarabine in adult participants with refractory or recurrent AML. In this study MK-8242 was administered at 30-250mg (p.o; QD) or 120-250mg (p.o; BID), for 28 days, 7 days/7 days off, and 210 or 300mg (p.o; BID), for 21 days, 7 days/14 days off. The study consisted of 26 patients, 5 of whom were discontinued due to AE and 7 of whom died. The study showed that the 7 day/14 day off regimen has more favorable safety than the 7 day/7 day off regimen. NCT01463696 is intended to evaluate the safety and pharmacokinetic profile of MK-8242 in patients with advanced solid tumors. In this study, drug doses were increased in part 1 to determine MTD, and MTD was confirmed in part 2 and recommended phase 2 dose (RPTD) was determined. Finally, 47 patients were enrolled in the study and treated with MK-8242 at 8 levels of doses of 60-500 mg. The results show that MK-8242 activates the p53 pathway with acceptable tolerability profile at 400mg (BID).

MDM2 inhibitor BI 907828 is an oral inhibitor of mouse double-minute 2 (MDM 2) and has potential anti-tumor activity. Upon oral administration, BI 907828 binds to MDM2 protein and prevents it from binding to the transcriptional activation domain of tumor suppressor protein p 53. By preventing MDM2-p53 interactions, the transcriptional activity of p53 is restored. This resulted in the induction of p53 mediated apoptosis in tumor cells. The pharmacokinetic properties of BI 907828 allow for more optimized dosing and dosing regimens than currently available MDM2 inhibitors, which may reduce myelosuppression, which is the dose-limiting toxicity of the hit target (on-target) of such inhibitors.

NVP-CGM097

Is a highly potent and selective MDM2 inhibitor, and has a Ki value of 1.3nM for hMDM2 in the TR-FRET assay. It binds to the p53 binding site of the Mdm2 protein, disrupting the interaction between the two proteins, resulting in activation of the p53 pathway.

Mirad Meptan

Is an oral MDM2 (mouse double-micro-body 2) antagonist with potential anti-tumor activity. Upon oral administration, mi Lade maytansinoid binds and prevents the binding of MDM2 protein to the transcriptional activation domain of tumor suppressor protein p 53. By preventing this MDM2-p53 interaction, proteasome-mediated enzymatic degradation of p53 is inhibited and the transcriptional activity of p53 is restored. This results in restoration of p53 signaling and in p 53-mediated induction of tumor cell apoptosis. MDM2, a negative regulator of zinc finger protein and p53 pathway, is overexpressed in cancer cells; it is associated with cancer cell proliferation and survival.

Salts of any of the above compounds are within the scope of the invention.

As used herein, MDM2 inhibitors may be compounds disclosed in the following applications: U.S. application Ser. No. 11/626,324, U.S. application publication No. 2008/0015194; U.S. non-provisional application Ser. No. 12/986,146; international application No. PCT/US11/20414, publication No. WO 2011/085126; or International application No. PCT/US11/20418, publication No. WO 2011/085129; each of which is incorporated herein by reference.

The MDM2 inhibitor may be a compound as disclosed in Vassilev 2006Trends in Molecular Medicine 13 (1), 23-31. For example, the MDM2 inhibitor may be nutlin (e.g., a cis-imidazole compound, such as nutlin-3 a); benzodiazepines (benzodiazepines) as disclosed in Grasberger et al, 2005J Med Chem 48,909-912; RITA compounds as disclosed in isaeva et al 2004Nat Med 10,1321-1328; spirooxindole compounds as disclosed in Ding et al, 2005J Am Chem Soc 127,10130-10131 and Ding et al, 2006J Med Chem 49,3432-3435; or a quinnol compound as disclosed by Lu et al, 2006J Med Chem 49,3759-3762. As another example, the MDM2 inhibitor may be, for example, chene2003nat. Rev. Cancer 3,102-109; fotouhi and Graves 2005Curr Top Med Chem 5,159-165; or a compound disclosed in Vassilev 2005J Med Chem 48,4491-4499.

An important advantage of the MDM2 inhibitors of the present invention is that MDM2 inhibition promotes cytotoxicity and longevity of donor T cells.

In embodiments, MDM2 inhibition can affect the phenotype of allogeneic T cells in the patient, resulting in increased cytotoxicity and longevity. For example, MDM2 inhibition may result in allogeneic T cells upregulating the expression of Bcl-2-receptor and IL 7-receptor (DE 127), markers associated with longevity. Furthermore, in the context of the present invention, increased expression of cytotoxic markers, such as perforin, CD107a, IFN- γ, TNF and CD69, by cd8+ allogeneic T cells can be observed when MDM2 inhibitors inhibit MDM 2.

Cytotoxic T cells (also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, cd8+ T cells, or killer T cells) are a type of T lymphocyte (a leukocyte) that kills cancer cells, infected (particularly virally infected) cells, or otherwise damaged cells. Most cytotoxic T cells express a T Cell Receptor (TCR) that recognizes a specific antigen. An antigen is a molecule capable of stimulating an immune response and is typically produced by a cancer cell or virus. Antigens within the cell bind to and are carried by MHC class I molecules to the cell surface where they can be recognized by T cells. If the TCR is specific for the antigen, it binds to a complex of an MHC class I molecule and antigen, and the T cell destroys the cell. In order for a TCR to bind to MHC class I molecules, the former must be accompanied by a glycoprotein known as CD8, which binds to a constant portion of MHC class I molecules. Thus, these T cells are referred to as cd8+ T cells. The affinity between CD8 and MHC molecules keeps TC cells and target cells tightly bound together during antigen-specific activation. Cd8+ T cells, once activated, are recognized as TC cells and are generally classified as having a predetermined cytotoxic effect in the immune system. Cd8+ T cells can also produce some cytokines.

Administration of an MDM2 inhibitor may induce up-regulation and increased expression of TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1), TRAIL-R2, human Leukocyte Antigen (HLA) class I molecules, and HLA class II molecules on cancer cells in a patient. TNF-related apoptosis-inducing ligands (TRAIL) are proteins that function as ligands for the induced process of cell death, known as apoptosis. TRAIL is a cytokine produced and secreted by most normal tissue cells. It causes apoptosis mainly in tumor cells by binding to certain death receptors TRAIL-R1 or TRAIL-R2. TRAIL is also named CD253 (cluster of differentiation 253) and TNFSF10 (tumor necrosis factor (ligand)) superfamily, member 10.

TNF-related apoptosis-inducing ligands (TRAIL) and 5 cell receptors thereof constitute one of three death receptor/ligand systems that have been shown to regulate the inter-cell apoptotic response in the immune system. TRAIL/TRAIL receptor systems have been shown to have immunosuppressive, immunomodulatory, proviral or antiviral and tumor immunomonitoring functions in different systems for antigen or tumor challenge. TRAIL binds to two apoptosis-inducing receptors, TRAIL-R1 (DR 4) and TRAIL-R2 (DR 5), and two other cell-binding receptors incapable of transmitting apoptosis signals, TRAIL-R3 (LIT, dcR 1) and TRAIL-R4 (trunk, dcR 2), sometimes referred to as decoy receptors. The initial step in TRAIL induction of apoptosis is ligand binding to TRAIL-R1 or TRAIL-R2. Thereby trimerizing the receptor and assembling the Death Inducing Signaling Complex (DISC). The adapter molecule Fas-associated death domain (FADD) is transferred to the DISC where it interacts with the intracellular Death Domain (DD) of the receptor. Through its second functional domain, the Death Effector Domain (DED) FADD recruits zymogens (procaspases) 8 and 10 to the DISC where they are autocatalytically active. This activation marks the onset of caspase-dependent signaling cascade amplification. Complete activation of effector caspases results in cleavage of the target protein, fragmentation of DNA, and ultimately cell death. The functions of TRAIL and TRAIL-R1 and TRAIL-R2 have been described in the art, for example by Falschlehner et al (immunology.2009 Jun;127 (2): 145-154).

Surprisingly, it was found that in the context of the present invention, MDM2 inhibition enhances TRAIL-R1/R2 expression on cancer cells, which in the context of the present invention is at least partially necessary to mediate the cytotoxic effects of allogeneic T cells, as deletion of TRAIL on T cells results in a substantial reduction in killing.

Furthermore, it is entirely unexpected that MDM2 inhibition can up-regulate MHC proteins on cancer cells (e.g., leukemia cells, particularly AML cells), thereby enhancing their vulnerability to allogeneic T cells after HCT and allogeneic T cell transplantation.

The Major Histocompatibility Complex (MHC) is a large locus on vertebrate DNA that contains a set of closely linked polymorphic genes encoding cell surface proteins necessary for the adaptive immune system. The locus is named because it was found in post-implantation histocompatibility studies. Later studies revealed that tissue rejection due to incompatibility is an experimental artifact that masks the true function of MHC molecules to bind antigens derived from self-proteins or pathogens and present the antigens on the cell surface for recognition by appropriate T cells. MHC molecules mediate interactions of leukocytes with other leukocytes or somatic cells. MHC determines the compatibility of donors for organ transplants and susceptibility to autoimmune diseases by cross-reactive immunity.

MHC class I molecules are expressed in all nucleated cells, but also in platelets, and in virtually all cells except erythrocytes. It presents epitopes to killer T cells, also known as Cytotoxic T Lymphocytes (CTLs). In addition to the T Cell Receptor (TCR), CTLs express the CD8 receptor. When the CD8 receptor of a CTL interfaces with an MHC class I molecule, if the TCR of the CTL matches an epitope within the MHC class I molecule, the CTL triggers the cell to undergo programmed cell death by apoptosis. Thus, MHC class I helps to mediate cellular immunity, a primary means of addressing intracellular pathogens (e.g., viruses and some bacteria, including bacterial type L, bacterial mycoplasma and bacterial rickettsia). In humans, MHC class I includes HLA-A, HLA-B and HLA-C molecules.

MHC class II can be conditionally expressed by all cell types, but is typically only present on "professional" Antigen Presenting Cells (APCs): macrophages, B cells, especially Dendritic Cells (DCs). APCs ingest antigen proteins, process antigens, and return their molecular moiety, a moiety called an epitope, and display it on the surface of APCs coupled within MHC class II molecules (antigen presentation). On the cell surface, epitopes can be recognized by immune structures such as T Cell Receptors (TCRs). The molecular region that binds an epitope is paratope. Helper T cells have CD4 receptors and TCRs on their surfaces. When the CD4 molecule of naive helper T cells is docked to the MHC class II molecule of APC, their TCR can contact and bind to the epitope coupled within the MHC class II molecule. This event triggers naive T cells (naive T cells). Naive helper T cells (Th 0) are polarized to memory Th cells or effector Th cells with phenotype of type 1 (Th 1), type 2 (Th 2), type 17 (Th 17) or regulatory/suppression (Treg) according to the balance of cytokines secreted by APCs in the local environment, i.e. microenvironment, as identified so far, are terminal differentiation of Th cells. Thus, MHC class II mediates immune tolerance to an antigen, or if APCs polarize Th0 cells primarily into Treg cells. Polarization during initial antigen exposure is critical in determining a variety of chronic diseases such as inflammatory bowel disease and asthma by shifting the coordinated immune response of memory Th cells when memory recall is triggered by re-exposure to similar antigens. B cells express MHC class II to present antigens to Th0, but these activated B cells secrete soluble immunoglobulins, antibody molecules that mediate fluid immunity when their B cell receptors bind to matched epitopes (not interactions mediated by MHC). Class II MHC molecules are also heterodimers, with both the α and β subunit genes being polymorphic and located in MHC class II regions. The peptide binding groove of an MHC-II molecule is formed by the N-terminal domains of the two subunits of heterodimers α1 and β1, unlike an MHC-I molecule, where two domains of the same chain are involved. In addition, both subunits of MHC-II contain transmembrane helices and immunoglobulin domains α2 or β2 that are recognized by the CD4 co-receptor. Thus, since different lymphocytes express different T Cell Receptor (TCR) co-receptors, MHC chaperone (chaperone) type lymphocytes can bind a given antigen with high affinity.

The Human Leukocyte Antigen (HLA) system or complex is a group of related proteins encoded by the gene complex of the human Major Histocompatibility Complex (MHC). HLA (both HLA class I groups) corresponding to MHC class I (A, B and C) present peptides from the cell interior. For example, if a cell is infected with a virus, the HLA system brings the viral fragment to the cell surface so that the cell can be destroyed by the immune system. These peptides are produced by digestion proteins that break down in the proteasome. Typically, these specific peptides are small polymers of about 8-10 amino acids in length. The foreign antigen presented by MHC class I attracts T lymphocytes called killer T cells (also called CD8 positive or cytotoxic T cells), which destroy the cells. Some new work has suggested that antigens longer than 10 amino acids (11-14 amino acids) can be presented on MHC I, eliciting a cytotoxic T cell response. [3] MHC class I proteins associate with β2-microglobulin, unlike HLA proteins encoded by genes on chromosome 15.

HLA corresponding to MHC class II (DP, DM, DO, DQ and DR) presents antigens from outside the cell to T lymphocytes. These specific antigens stimulate proliferation of T helper cells (also known as CD 4-positive T cells), which in turn stimulate antibody-producing B cells to produce antibodies against the specific antigen. Autoantigens are inhibited by regulatory T cells.

Export protein 1 (XPO 1), also known as chromosome maintenance protein 1 (CRM 1), is a eukaryotic protein that mediates nuclear export of proteins, rRNA, snRNA and some mRNAs. Export protein 1 mediates leucine-rich Nuclear Export Signal (NES) -dependent protein transport and specifically mediates nuclear export of Rev and UsnRNA. It is involved in the control of several cellular processes by controlling the localization of cyclin B, MAPK and MAPKAP kinase 2, and it also regulates NFAT and AP-1. Furthermore, it has been shown to interact with p53 and mediate its export from the nucleus, thereby reducing the expression of genes under the control of p53 such as those encoding TRAIL-R1 and-R2 and MHC-II.

XPO1 is also upregulated in many malignancies and is associated with poor prognosis. Their inhibition has become the target of therapy, and therefore, selective inhibitors of nuclear transport (SINE) compounds have been developed as a new class of anticancer agents. The most well known SINE agent is plug Li Nisuo (selinexor) (KPT-330) and has been widely tested in phase I and II clinical trials in solid tumors and hematological malignancies.

A selective inhibitor of nuclear export (a SINE or a SINE compound) is a drug that blocks exporter 1 (XPO 1 or CRM 1), a protein involved in transport from the nucleus to the cytoplasm. This causes cell cycle arrest and cell death by apoptosis. Thus, SINE compounds are of interest as anticancer drugs; some are under development, one (plug Li Nisuo) has been approved as a drug for the last resort to for the treatment of multiple myeloma. The protonuclear export inhibitor is leptomycin B, a natural product and a secondary metabolite of bacteria of the genus streptomyces. SINE includes, for example, KPT-8602, KPT-185, KPT-276KPT-127, KPT-205, and KPT-227 in addition to KPT-330. XPO-1 inhibition for therapeutic purposes has been reviewed in the literature, e.g., parikh et al (J Hematol Oncol.2014; 7:78).

As used herein, a pharmaceutical composition for administration to a subject may comprise at least one additional pharmaceutically acceptable additive, such as a carrier, thickener, diluent, buffer, preservative, surfactant, and the like, in addition to the selected molecule. The pharmaceutical composition may also comprise one or more additional active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. Pharmaceutically acceptable carriers useful in these formulations are conventional. Compositions and formulations suitable for drug delivery of the compounds disclosed herein are described by Remington's Pharmaceutical Sciences, mack Publishing co., easton, PA, 19 th edition (1995) of martin.

Generally, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically contain an injectable fluid which includes pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol and the like as vehicles (vehicles). For solid compositions (e.g., in the form of a powder, pill, tablet, or capsule), conventional non-toxic solid carriers may include, for example, medical grade mannitol, lactose, starch, or magnesium stearate. In addition to the bio-neutral carrier, the pharmaceutical composition to be administered may contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

According to various methods of treatment of the present disclosure, a compound may be delivered to a subject in a manner consistent with conventional methods associated with management of a disorder sought to be treated or prevented. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of a compound and/or other bioactive agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit and/or ameliorate a selected disease or disorder or one or more symptoms thereof.

"administration (administration of)" and "administering" a compound or product is understood to mean providing a compound, a prodrug of a compound, or a pharmaceutical composition as described herein. The compound or composition may be administered (e.g., intravenously) to the subject by another person, or it may be administered by the subject itself (e.g., a tablet).

Any reference herein to a compound for use as a medicament for treating a medical condition (medical condition) also relates to a method of treating the medical condition comprising administering the compound or a composition comprising the compound to a subject in need thereof, or to the use of the compound, a composition comprising the compound in the treatment of the medical condition.

The physician can vary the dosage to maintain the desired concentration at the target site (e.g., lung, bone marrow, or systemic circulation). Higher or lower concentrations may be selected based on the mode of delivery, e.g., transdermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. The dosage may also be adjusted based on the release rate of the formulation being administered, such as by intrapulmonary spraying of powders, slow-release oral and injectable granules, or transdermal delivery formulations, etc.

The invention also relates to methods of treating a subject as disclosed herein. The method of treatment preferably comprises administering to a subject in need thereof a therapeutically effective amount of a compound disclosed herein and possibly additional compounds or products.

In the context of the present invention, the term "pharmaceutical" refers to a drug, pharmaceutical or pharmaceutical product for diagnosing, curing, treating or preventing a disease. It refers to any substance or combination of substances that has the property of treating or preventing a disease. The term includes any substance or combination of substances that can be used or administered to restore, correct or alter physiological function, or make a medical diagnosis by exerting pharmacological, immunological or metabolic effects. The term pharmaceutical includes biopharmaceuticals, small molecule drugs, or other physical materials that affect physiological processes.

The MDM2 inhibitors and possibly other compounds according to the invention as described herein may comprise different types of carriers, depending on whether they are administered in solid, liquid or aerosol form, and whether they need to be sterile for the route of administration such as injection. The invention may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, subcutaneously, subconjunctival, intracapsular, mucosal, intracardiac, intraumbilical, intraocular, oral, topical, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, direct local infusion of bathing target cells, via catheters, via lavage, in cream form, in lipid compositions (e.g., liposomes) or by other methods known to those of ordinary skill in the art or any combination of the foregoing (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, mack printing company, 1990, incorporated herein by reference).

In the context of the present invention, the term "cancer therapy" refers to any type of cancer treatment, including but not limited to surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy, cell therapy, cancer immunotherapy, monoclonal antibody therapy. Administration of MDM2 inhibitors as described herein may be embedded in a broader cancer treatment strategy.

Administration of the MDM2 inhibitor may be combined with one or more other cancer therapies. In the context of the present invention, the term "combination" means that an individual receiving a compound according to the present invention also receives other cancer therapies (which do not necessarily occur simultaneously) combined in a single pharmacological composition or via the same route of administration. Thus, "combination" refers to treating an individual with cancer with more than one cancer therapy. Combination administration encompasses simultaneous, co-therapy, or combination therapy (joint treatment), wherein treatments may occur within minutes of each other, within the same hour, within the same day, within the same week, or within the same month.

Cancer therapies in the sense of the present invention include, but are not limited to, radiation therapy and chemotherapy, and act by overwhelming the ability of cells to repair DNA damage, resulting in cell death.

Chemotherapy, as used herein, refers to a category of cancer treatment that uses one or more anticancer drugs (chemotherapeutic agents) as part of a standardized chemotherapy regimen. Chemotherapy may be administered for curative purposes (which almost always involve a combination of drugs), or it may be aimed at prolonging life or reducing symptoms (palliative chemotherapy). Chemotherapy is one of the main categories of medical oncology (the medical disciplines dedicated to cancer drug therapies). Chemotherapeutic agents (chemotherapeutic agent) are used to treat cancer and are administered on a regimen of one or more cycles, combining two or more agents over a period of days to weeks. Such agents are toxic to cells with high proliferation rates, for example to the cancer itself, but also to the GI tract (causing nausea and vomiting), bone marrow (causing various cytopenias) and hair (causing alopecia).

Chemotherapeutic agents include, but are not limited to, actinomycin, all-trans retinoic acid, azacytidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, nitrogen mustard, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, pentarubicin, vinblastine, vincristine, vindesine, and vinorelbine.

In the context of the present invention, radiation or radiotherapy refers to treatment methods using ionizing or ultraviolet-visible (UV/Vis) radiation, typically as part of a cancer treatment to control or kill malignant cells such as cancer cells or tumor cells. Radiation therapy may be curative in many types of cancers if the cancer is located in a region of the body. It can also be used as part of adjuvant therapy to prevent postoperative tumor recurrence, thereby removing primary malignancy (e.g., early stage breast cancer). Radiation therapy is synergistic with chemotherapy and can be used to treat susceptible cancers before, during, and after chemotherapy. Radiation therapy is commonly used in cancerous tumors because of its ability to control cell growth. Ionizing radiation works by destroying the DNA of cancerous tissue leading to cell death. Radiation therapy may be used systemically or locally.

Radiation therapy works by destroying the DNA of cancer cells. This DNA damage is caused by one of two types of energy, photons or charged particles. Such damage is the direct or indirect ionization of atoms constituting the DNA strand. Indirect ionization occurs due to ionization of water, leading to the formation of free radicals, including hydroxyl radicals, which subsequently damage the DNA. In photon therapy, most of the radiation effects are mediated by free radicals. Cells have mechanisms to repair single-stranded and double-stranded DNA damage. However, double-stranded DNA breaks are more difficult to repair and can lead to significant chromosomal abnormalities and gene deletions. Targeting double strand breaks increases the likelihood that the cell will undergo cell death.

The amount of radiation used in photon radiation therapy is measured in gray scale (Gy) and varies depending on the type and stage of cancer being treated. For the treatment of cases, typical doses for solid epithelial tumors are 60-80Gy, whereas lymphomas are treated with 20-40 Gy. The prophylactic (adjuvant) dose is typically about 45-60Gy per 1.8-2Gy (fraction) for breast cancer, head and neck cancer.

Different types of radiation therapies are known, such as external beam radiation therapies, including conventional external beam radiation therapy, stereotactic radiation (radiosurgery), virtual simulation, 3-dimensional conformal radiation therapy, and intensity modulated radiation therapy, intensity Modulated Radiation Therapy (IMRT), volume Modulated Arc Therapy (VMAT), particle therapy, auger therapy, brachytherapy, intra-operative radiation therapy, radioisotope therapy, and deep inhalation breath hold.

External beam radiation therapy includes X-rays, gamma rays, and charged particles, and may be applied at low or high dose rates depending on the overall treatment method.

The radioactive material may be bound to one or more monoclonal antibodies during internal radiation therapy. For example, radioiodine may be used in thyroid malignancy. Brachytherapy of either a High Dose Regimen (HDR) or a Low Dose Regimen (LDR) can be used in combination with IR for prostate cancer.

According to the present invention, DNA damage-inducing chemotherapy includes the administration of chemotherapeutic agents including, but not limited to, anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone; topoisomerase I inhibitors such as irinotecan (CPT-11) and topotecan; topoisomerase II inhibitors including etoposide, teniposide and tafluporide (Tafluposide); platinum-based agents such as carboplatin, cisplatin, and oxaliplatin; and other chemotherapies such as bleomycin.

The present disclosure also includes kits, packages, and multi-container units containing the pharmaceutical compositions, active ingredients, and/or means for administering the pharmaceutical compositions, active ingredients described herein for the prevention and treatment of diseases and other symptoms in mammalian subjects.

Drawings

The invention is further described by the following figures. These examples are not intended to limit the scope of the invention, but rather to represent preferred embodiments of aspects of the invention, which are provided to better illustrate the invention described herein.

Brief description of the drawings:

fig. 1: MDM2 inhibition improves AML survival in multiple GVL mouse models

(a) The percent survival of BALB/C recipient mice after transfer of AML WEHI-3B cells (BALB/C background) and allogeneic C57BL/6BM is shown. As shown, the mice were injected with additional allogeneic T cells (C57 BL/6) and/or treated with vehicle or MDM2 inhibitor RG-7112. N=9-10 independent animals per group are shown and p-values are calculated using a double sided Mantel-Cox test.

(b) Showing transfer of AML ^{MLL-PTD FLT3-ITD} Percent survival of cells (C57 BL/6 background) and C57BL/6 receptor mice after allogeneic BALB/cBM. As shown, mice are injected with additional allogeneic T cells (BALB/c) and/or treated with vehicle or MDM2 inhibitor RG-7112. N=10 biologically independent animals from both experiments are shown and p-values are calculated using a two-sided Mantel-Cox test.

(c) Shows Rag2 after transfer of human OCI-AML-3 cells ^–/– Il2rγ ^–/– Percent survival of recipient mice. As shown, mice are injected with additional human T cells (peripheral blood isolated from healthy donors) and/or treated with vehicle or MDM2 inhibitor RG-7112. N=12 biologically independent animals from three experiments are shown and p-values are calculated using a two-sided Mantel-Cox test.

(d) The percentage of specific lysis of isolated CD3/28 and IL-2 expanded human T cells in contact with OCI-AML3 cells is shown. OCI-AML3 cells were pretreated with DMSO or MDM2 inhibitor RG-7112 and the E: T ratio of effector (T cells) to target (OCI-AML 3 cells) was varied as shown between 10:1 and 1:1. A representative experiment of three independent experiments is shown.

(e) Representative western blots showed activation of caspase 3 and loading control (β -actin) in OCI-AML3 cells. OCI-AML3 cells exposed to DMSO or RG-7112 (1. Mu.M) were co-cultured with activated T cells at a 10:1 E:T ratio for 4 hours.

(f) The bar graph shows the ratio of cleaved caspase 3 normalized to β -actin to pro-caspase 3. These values were normalized to the T cell only group (set to "1").

(g) Microarray-based analysis of TNFRSF10A and TNFRSF10B expression levels in OCI-AML3 cells after 24 hours of treatment with DMSO, RG-7112 (1 μm), or HDM-201 (200 nM) showed tiling from Robust Multichip Average (RMA) signal values, n=6 biologically independent samples/group.

(h) The graph shows the fold change in MFI of TRAIL-R1 expression on OCI-AML3 cells after 72 hours of treatment with the indicated concentration of MDM2 inhibitor RG-7112, which is the mean ± SEM from n=5 independent experiments. P-values were calculated using a double sided Student unpaired t-test.

(i) The graph shows the fold change in MFI of TRAIL-R2 expression on OCI-AML3 cells after 72 hours of treatment with the indicated concentration of MDM2 inhibitor RG-7112, which is the mean ± SEM from n=5 independent experiments. P-values were calculated using a double sided Student unpaired t-test.

(j, k) the graph shows that OCI-AML3 (p 53) after 72 hours of treatment with MDM2 inhibitor RG-7112 at the indicated concentrations ^+/+ ) Or p53 knockout (p 53) ^-/- ) Fold change in MFI expressed by TRAIL-R1 (j) or TRAIL-R2 (k) on OCI-AML3 cells, which is mean ± SEM from n=4 independent experiments. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(l, M) ChIP-qPCR analysis in OCI-AML3 cells treated with DMSO or 2 μm RG-7112 for 12 hours to detect p53 binding to the promoters of TRAIL-R1 (TNFRSF 10A) (l) and TRAIL-R2 (TNFRSF 10B) (M). Data are expressed as input percentages and represent three experiments; error bars s.e.m. from triplicate technical replicates. And N.D: no detection was made.

Fig. 2: MDM2 inhibition enhances TRAIL-R1/2 expression in a p 53-dependent manner

(a) Showing transfer of AML ^{MLL-PTD FLT3-ITD} Percent survival of cells (C57 BL/6 background) and C57BL/6 receptor mice after allogeneic BALB/cBM. Mice were injected with additional allogeneic T cells (BALB/c) and treated with MDM2 inhibitor RG-7112 and the indicated anti-TRAIL-antibody or IgG isotype. N=10 independent animals from 2 experiments are shown and p-values are calculated using a double sided Mantel-Cox test.

(b) Showing transfer of AML ^{MLL-PTD FLT3-ITD} Percent survival of cells (C57 BL/6 background) and C57BL/6 receptor mice after allogeneic BALB/cBM. Mice were injected with additional allogeneic T cells (BALB/c), either WT T cells or TRAIL ^-/- T cells. N=10 independent animals from 2 experiments are shown and p-values are calculated using a double sided Mantel-Cox test.

(c) Western blot shows activation of caspase 3, caspase 9 and loading control (β -actin) in OCI-AML3 cells. Activated T cells were pretreated with 10. Mu.g/ml anti-TRAIL, neutralizing antibody or IgG control for 1 hour and co-cultured with OCI-AML3 cells exposed to DMSO or RG-7112 (1. Mu.M) at a 10:1 E:T ratio for 4 hours.

(d) Quantification of the ratio of cleaved caspase 3/total caspase 3 normalized to isotype control. Each data point represents an independent biological repeat.

(e) Quantification of the ratio of cleaved caspase 9/total caspase 9 normalized to isotype control. Each data point represents an independent biological repeat.

(f) Rag2 receiving WT OCI-AML cells or TRAIL-R2 CRISPR-Cas knockout OCI-AML cells ^–/– Il2rγ ^–/– Survival rate of mice. Mice were additionally injected with primary human T cells isolated from healthy donors and treated with vehicle or MDM2 inhibitor RG-7112.N=10 animals from two independent experiments are shown and p-values are calculated using a double sided Mantel-Cox test.

(g) Bar graphs show that WT or TRAIL-R2 CRISPR-Cas knocks out OCI-AML3 cells (TRAIL-R2 ^-/- ) The cells were incubated with 1. Mu.M MDM2 inhibitor RG7112 as shown. After 48 hours, hTRAIL (TNFSF 10) was added at defined concentrations for 24 hours as shown. The viability of AML cells was measured by flow cytometry. Mean ± SEM of triplicate are shown. P-values were calculated using a double sided Student unpaired t-test.

(h) BALB/c mice carrying WEHI-3B leukemia isolated CD8 from spleen on day 12 post-allogeneic HCT ⁺ Extracellular acidification Rate of T cells (ECAR) the BALB/C mice have undergone allogeneic HCT with C57BL/6BM plus allogeneic C57BL/6T cells. As shown, recipient mice were treated with either the vehicle or the MDM2 inhibitor RG-7112. For each repetition, normalization of ECAR baseline values was performed. Mean ± SEM from n=4 biologically independent replicates, each replicate was generated by pooling spleens from two mice. P values were calculated using a double sided unpaired Student t test.

(i) CD8 isolated from BMT receptor as depicted in Panel h ⁺ Glycolysis (calculated as the difference between ECAR and basal ECAR after glucose injection) and glycolytic capacity (calculated as the difference between ECAR and basal ECAR after oligomycin injection) of T cells. n=average of 4 biologically independent replicates ± SEM, each replicate was generated by pooling spleens from two mice. P values were calculated using a double sided unpaired Student t test.

(j) As depicted in Panel h, CD8 isolated from BMT receptor was labeled ex vivo ⁺ After T cells, U- ¹³ The fractional contribution of C-glucose to glycolytic intermediates. Each dot represents a single mouse. P values, ns were calculated using a double sided unpaired Student t test: is not significant. The pathway schematic was created using a biorender.

Fig. 3: MDM2 inhibition promotes cytotoxicity and longevity of donor T cells

(a-h) scatter plots and representative histograms show that the vehicle was used in conjunction with C57BL/6BM plus allogeneic C57BL/6T cell transplantationOr MDM2 inhibitor RG-7112 treated BALB/c mice bearing WEHI-3B leukemia isolated CD8 from spleen on day 12 post allogeneic HCT ⁺ Perforin (a, b), CD107a (c, d), IFN- α (e, f), TNF- α (g, h) expression in T cells. Mean ± SEM of n=14-19 biologically independent animals from each group of 2 experiments are shown, and p-values are calculated using a double sided Mann-Whitney U test.

(i) Shows the use of allogeneic BALB/cBM to transfer AML ^{MLL-PTD FLT3-ITD} Percent survival of cells (C57 BL/6 background) and C57BL/6 receptor mice after BMT. Mice were injected with additional allogeneic T cells (BALB/c) on day 2 post-BMT. When the CD 8T cells or NK cells are depleted. N=10 independent animals from 2 experiments are shown and p-values are calculated using double sided Mantel-Cox.

(j) Showing transfer of AML ^{MLL-PTD FLT3-ITD} Percent survival of cells (C57 BL/6 background) and C57BL/6 receptor mice after allogeneic BALB/cBM. Mice were injected with additional allogeneic T cells (BALB/c) from previously challenged and treated (MDM 2 inhibitor or vehicle) mice. N=10 independent animals from 2 experiments are shown and p-values are calculated using a double sided Mantel-Cox test.

(k) UMAP shows FlowSOM-guided artificial meta-clustering (A, upper panel), and heat-panels show median marker expression from spleen viable CD45+ cells from BALB/c mice carrying allograft leukemia (lower panel).

(l) UMAP shows FlowSOM-guided manual meta-clustering (A, upper panel), heat-maps show median marker expression from donor-derived (H-2 kb+) TCRb+CD8+ T cells from BALB/c mice carrying allograft leukemia treated with either RG-7112 or vehicle as indicated (lower panel).

(m) quantification of donor-derived (H-2 kb+) TCRb+CD8+CD27+TIM3+ T cells from BALB/c mice bearing allograft leukemia, mice were treated with RG-7112 or vehicle as shown.

Fig. 4: MDM2 inhibition in primary human AML cells results in TRAIL-1/2 expression

(a) The figure shows hTRAIL-R1 mRNA expression levels in primary human AML cells normalized to hGapdh, as determined by qPCR, before or after 12 hours of in vitro treatment with RG-7112 (2 μm). Each data point represents a single sample of an individual patient. Experiments were performed independently and the results were pooled (mean ± s.e.m.).

(b) The figure shows representative quantification of hTRAIL-R1 mRNA levels of primary AML blasts from patient-derived PBMCs after 12 hours of in vitro treatment with RG-7112 (0.5, 1 and 2 μm) at different concentrations.

(c) The figure shows hTRAIL-R2 mRNA expression levels in primary human AML cells normalized to hGapdh, as determined by qPCR, before or after 12 hours of in vitro treatment with RG-7112 (2 μm). Each data point represents a single sample of an individual patient. Experiments were performed independently and the results were pooled (mean ± s.e.m.).

(d) The figure shows representative quantification of hTRAIL-R2 mRNA levels of primary AML blasts from patient-derived PBMCs after 12 hours of treatment in vitro with RG-7112 (0.5, 1 and 2 μm) at different concentrations.

(e) Shows Rag2 after transfer of primary human AML cells ^–/– Il2rγ ^–/– Percent survival of recipient mice (patient # 56). As shown, mice were injected with additional human T cells (peripheral blood isolated from HLA-mismatched healthy donors) and/or treated with vehicle or MDM2 inhibitor RG-7112. N=10 independent animals are shown and p-values are calculated using a double sided Mantel-Cox test.

(f) Shows the effect of the expression of the gene in a metastatic human WT or p53 knockout (p 53 ^-/- ) Rag2 after OCI-AML-3 cells ^–/– Il2rγ ^–/– Percent survival of recipient mice. As shown, mice were injected with additional human T cells (peripheral blood isolated from HLA-mismatched healthy donors) and/or treated with vehicle or MDM2 inhibitor RG-7112. N=10 biologically independent animals from both experiments are shown and p-values are calculated using the double sided Mantel-Cox test.

(g) Representative western blots showed caspase 8, caspase 3, PARP and loading control (β -actin) in human OCI-AML3 cells. OCI-AML3 cells exposed to DMSO or RG-7112 (1. Mu.M) were co-cultured with activated T cells at a 10:1 E:T ratio for 4 hours. These values were normalized to β -actin.

(h, i) representative flow cytometry histograms (h) and fold change histograms (i) show the Mean Fluorescence Intensity (MFI) of HLA-C expression on OCI-AML3 cells after 72 hours of treatment with the indicated concentrations of MDM2 inhibitor RG-7112. Bar graphs show mean ± SEM from n=5-6 independent experiments. P-values were calculated using a double sided Student unpaired t-test.

(j, k) representative flow cytometry histograms (j) and fold change histograms (k) show the Mean Fluorescence Intensity (MFI) of HLA-DR expression on OCI-AML3 cells after 72hours of treatment with the indicated concentrations of MDM2 inhibitor RG-7112. Bar graphs show mean ± SEM from n=5-6 independent experiments. P-values were calculated using a double sided Student unpaired t-test.

(l, m) the graph shows fold changes in MFI of HLA-C. (l) HLA-DR (m) expression on OCI-AML3 (p 53) ^+/+ )or p53 knockdown(p53 ^-/- ) OCI-AML3 cells after treatment with RG-7112 (2 μm) for 72hours as mean±sem from n=4 independent experiments the MFI of control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(n) cumulative HLA-DR (MHC-II) levels of primary AML patient blast cells were determined by flow cytometry after 48 hours of in vitro treatment with RG-7112 (2 μm) and shown as MFI for n=11 biologically independent patients. The MFI of HLA-DR (MHC-II) from control treated cells was set to 1.0. P-values were calculated using a two-sided Wilcoxon paired symbol rank test (Wilcoxon matched-pairs signed rank test) and are shown in the figure.

(o) representative histogram shows that after 48 hours of in vitro treatment with MDM2 inhibitor RG-7112 at the indicated concentrations, the MFI of HLA-DR expression on primary AML blast cells of patients is the mean.+ -. SEM from one experiment performed in triplicate. MFI from control treated cells was set to 1.0 and p-values were calculated using a double sided Student unpaired t-test.

Fig. 5: GVHD histopathological scoring

(a-C) scatter plots show histopathological scores for (a) liver, (b) colon, (C) small intestine from C57BL/6 mice receiving BALB/cBM and T cells and treated with vehicle or MDM2 inhibitor RG-7112 on day 12 post allogeneic HCT. The double sided Mann-Whitney U test was used (calculated P-values were not significant (n.s.)).

Fig. 6: inhibition of TRAIL-R1/R2mRNA and protein expression in human OCI-AML3 cells after MDM2 with RG7112 or HDM201

(a) Representative flow cytometry histograms show the Mean Fluorescence Intensity (MFI) of TRAIL-R1 expression on OCI-AML3 cells after 72 hours of treatment with the indicated concentrations of MDM2 inhibitor RG-7112. One of 5 independent biological repeats is shown.

(b) Representative flow cytometry histograms show the Mean Fluorescence Intensity (MFI) of TRAIL-R2 expression on OCI-AML3 cells after 72 hours of treatment with the indicated concentrations of MDM2 inhibitor RG-7112. One of 5 independent biological repeats is shown.

(c-f) the graph shows the fold change in human TRAIL-R1 (htrilr 1) RNA and htrilr 2RNA in OCI-AML3 cells after 6 hours (c, d) or 12 hours (e, f) treatment with the indicated concentrations of MDM2 inhibitor RG-7112, which is the mean ± SEM from n=3 independent experiments for 2 technical replicates each. RNA from control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(g, i) representative flow cytometry histograms describe the Mean Fluorescence Intensity (MFI) of hTRAIL-R1 (g) and hTRAIL-R2 (i) expression on OCI-AML3 cells after 72 hours of treatment with the indicated concentrations of the MDM2 inhibitor HDM-201.

(h, j) the graph shows the fold change in MFI of TRAIL-R1 (h) and TRAIL-R2 (j) expression on OCI-AML3 cells after 72 hours of treatment with the indicated concentrations of MDM2 inhibitor HDM201, which is the mean ± SEM from n=5 independent experiments. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

Fig. 7: TRAIL-R mRNA and protein expression in mouse WEHI-3B cells

(a, B) the graph shows fold change in mouse TRAIL-R (mTRAIL-R) RNA and mTRAIL-R2RNA in WEHI-3B cells after 6h treatment with the indicated concentrations of MDM2 inhibitor RG-7112, which is the mean ± SEM of n=4 independent experiments. RNA of DMSO-treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(c, d) the graph shows fold changes in mouse TRAIL-R (mTRAIL-R) RNA and mTRAIL-R2RNA in WEHI-3B cells after 12 hours of treatment with the indicated concentrations of MDM2 inhibitor RG-7112, which is the mean ± SEM of n=4 independent experiments. RNA of DMSO-treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(e) Representative flow cytometry histograms describe the Mean Fluorescence Intensity (MFI) of TRAIL-R2 expression on WEHI-3B cells after 72 hours of treatment with the indicated concentrations of MDM2 inhibitor RG-7112.

(f) The graph shows the fold change in MFI of TRAIL-R2 expression on WEHI-3B cells after 72 hours of treatment with the indicated concentration of MDM2 inhibitor RG-7112, which is the mean ± SEM from n=5 independent experiments. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(g) Representative flow cytometry histograms describe the Mean Fluorescence Intensity (MFI) of TRAIL-R2 expression on WEHI-3B cells after 72 hours of treatment with the indicated concentrations of the MDM2 inhibitor HDM 201.

(h) The graph shows the fold change in MFI of TRAIL-R2 expression on WEHI-3B cells after 72 hours of treatment with the indicated concentration of MDM2 inhibitor HDM201, which is the mean ± SEM from n=5 independent experiments. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

Fig. 8: XI-006 (MDMX inhibitor) treatment resulted in increased TRAIL-R1/R2 expression.

(a) The graph shows the percentage of viable (dead living cell identification dye negative) OCI-AML3 cells treated with MDMX inhibitor XI-006 at the indicated concentrations for 72 hours, which is the mean ± SEM from n=7 independent experiments. P-values were calculated using a double sided Student unpaired t-test.

(b, c) the graph shows the fold change in MFI expressed by TRAIL-R1 (b) and TRAIL-R2 (c) on OCI-AML3 cells after 72 hours of treatment with MDMX inhibitor XI-006 at the concentrations indicated, which is the mean ± SEM from n=7 independent experiments. The MFI of DMSO-treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

Fig. 9: HDM201 (MDM 2 inhibitor) treatment increased TRAIL-R1/R2 expression on human OCI-AML3 cells in a p 53-dependent manner.

(a) Representative western blots (left panels) show the expression of MDM2, p53 and loading control (GAPDH) in WT OCI-AML3 cells or p53 knockout OCI-AML3 cells exposed to 1mg/ml doxorubicin for 4 hours, as shown. Right figure: the relative intensities of the protein bands of each group were quantified.

(b) Representative western blots showed the expression levels of MDM2, p53 and loading control (GAPDH) in OCI-AML3 cells exposed to RG-7112 at 1 μm for 4 hours (left panel).

(c, d) the graph shows wild-type (WT) OCI-AML3 or p53 knockout (p 53) after 72 hours of treatment with MDM2 inhibitor HDM201 at the indicated concentrations ^-/- ) Fold change in MFI expressed by TRAIL-R1 (c) and TRAIL-R2 (d) on OCI-AML3 cells, which is mean ± SEM from n=4 independent experiments. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(e) The graph shows the percentage of living cells. In the wild-type OCI-AML3 (WT) or p53 knockout (p 53) ^-/- ) In the following cases, OCI-AML3 was incubated with 1. Mu.M MDM2 inhibitor RG 7112. After 48 hours, hTRAIL (TNFSF 10) was added at defined concentrations for 24 hours as shown. Viability was measured by flow cytometry. Mean ± SEM of triplicate are shown. P-values were calculated using a double sided Student unpaired t-test.

Fig. 10: effects of TRAIL-R2 knockout efficacy and MDM2 inhibition in OCI-AML3 cells.

(a) Representative flow cytometry histograms describe the Mean Fluorescence Intensity (MFI) of hTRAIL-R2, hTRAIL-R1 and p53 expression on WT OCI-AML3 cells or when using CRISPR-Cas to knock out hTRAIL-R2. Treatment with the indicated concentrations of MDM2 inhibitor RG7112 was carried out for 72 hours.

(b) The graph shows fold change in MFI of TRAIL-R2 expression on WT or TRAIL-R2 CRISPR-Cas knockdown OCI-AML3 cells after 72 hours treatment with the indicated concentration of MDM2 inhibitor RG7112, which is the mean ± SEM from n=2 independent experiments. P-values were calculated using a double sided Student unpaired t-test.

(c) The viability of WT or TRAIL-R2 CRISPR-Cas knockdown OCI-AML3 cells after 24 hours of treatment with optimal concentration of hTRAIL (TNFSF 10) was measured by flow cytometry. Mean ± SEM of triplicate are shown. P-values were calculated using a double sided Student unpaired t-test.

Fig. 11: MDM2 inhibition increases the metabolic activity of alloreactive T cells

(a-c) enrichment of CD8 from spleen of allogeneic HCT receptor mice treated with MDM2 inhibitor ⁺ T cells. Polar metabolites were extracted from n=8 mice treated with vehicle and n=7 mice treated with MDM2 inhibitor and measured by LC-MS as described in the supplementation method. (a) Volcanic plot of 100 metabolites analyzed using the targeting method. P-values were calculated using unpaired double sided Student t-test. (b) Heat maps of 27 significantly regulated metabolites between "MDM2 inhibitor" and "carrier" (p<0.05). Color scale represents normalized concentration in each sample. (c) Absolute abundance of metabolites from pyrimidine biosynthetic pathway. Pathway scheme created with Biorender<0.05，**p<0.01。

Fig. 12: spleen H-2kb after MDM2 inhibition in leukemia bearing mice ⁺ CD8 ⁺ Gating strategy for CD69 expression on T cells and CD 8T cells.

(a) Flow cytometry shows the identification of donor-derived (H-2 kb) from the spleen of mice ⁺ )CD3 ⁺ CD8 ⁺ Gating strategy for T cells. The gating cells were singlet (single), H-2kb, which were living cells (dead living cells identified as negative for dye) ⁺ 、CD45 ⁺ 、CD3 ⁺ And CD8 ⁺ . Spleens were harvested from BALB/C mice subjected to TBI and injected with C57BL/6BM and WEHI-3B cells (d 0). Mice were infused with allogeneic donor T cells (d 2) and treated with 5 doses of RG-7112 every two days starting on day 3.

Fig. 13: phenotype of T cells isolated from mice treated with MDM2 inhibitors of allogeneic HCT.

(a) Representative flow cytometry histograms describe Mean Fluorescence Intensity (MFI) and scatter plots showing all living donors (H-2 kb) from leukemia bearing BALB/c mice ⁺ )CD8 ⁺ Fold change in MFI of CD69 of T cells, the BALB/c mice underwent allogeneic HCT and were treated with vehicle. Mean ± SEM of n=14/15 biologically independent mice from each group of 2 experiments are shown. The MFI of vehicle-treated leukemia-bearing mice was set to 1.0. The P-value was calculated using a two-sided Mann-Whitney U test.

(b) The scatter plot shows all living donors (H-2 kb) from BALB/c mice bearing allograft leukemia ⁺ )CD3 ⁺ CD8 of T cells ⁺ Percent of cells, the BALB/c mice were treated with RG-7112 or vehicle as indicated. Mean ± SEM of n=14/19 biologically independent mice from each group of 3 experiments are shown. The MFI of vehicle-treated leukemia-bearing mice was set to 1.0. The P-value was calculated using a two-sided Mann-Whitney U test. No CD 8T cell/all CD3T cell differences were detected.

Fig. 14: MDM2 inhibition promotes T cell cytotoxicity in naive mice

(a-d) flow cytometry analysis of spleen cells from naive C57BL/6 mice treated with 5 doses of RG-7112 or vehicle every two days. The time point analyzed was 1 day after the last treatment.

(a) The scatter plot shows all live donors (H-2 kb) from untreated blank C57BL/6 mice ⁺ )CD3 ⁺ CD8 of T cells ⁺ Percent of cells, the C57BL/6 mice were treated with RG-7112 or vehicle as indicated. Mean ± SEM of n=5/10 biologically independent mice from each group of 2 experiments are shown. The MFI of vehicle-treated leukemia-bearing mice was set to 1.0. The P-value was calculated using a two-sided Mann-Whitney U test.

(b-d) scatter plots show all living donors (H-2 kb) from untreated blank C57BL/6 mice treated with vehicle ⁺ )CD8 ⁺ CD3 ⁺ T cellFold change in MFI for CD107a (b), TNF (c), and CD69 (d). Mean ± SEM from 2 experiments, n=5/10 biologically independent mice per group, are shown. The MFI of vehicle-treated leukemia-bearing mice was set to 1.0. The P-value was calculated using a two-sided Mann-Whitney U test.

Fig. 15: CD8 ⁺ T cells or NK1.1 ⁺ Purity of BM grafts before and after cell depletion.

(a) Representative flow cytometry plots show CD8 removal by fluorescence activated cell sorting ⁺ BM purity before and after T cells. The sorted cells shown were used for BM CD8 ⁺ Survival experiments with depletion. Similar results were obtained in two independent experiments.

(b) Representative flow cytometry plots showed NK1.1 removal by fluorescence activated cell sorting ⁺ BM purity before and after cells. The sorted cells shown were used for viability experiments with BM NK cell depletion. Similar results were obtained in two independent experiments.

Fig. 16: CD3 for transfer in secondary receptors ⁺ CD8 ⁺ H-2kd ⁺ Purity of T cells

(a) Representative flow cytometry images showed spleen (all living cells) CD3 ⁺ H-2kd ⁺ CD8 ⁺ T cell purity, the cells were re-isolated from BALB/cBM, mouse AML ^{MLL-PTD/FLT3-ITD} C57BL/6 mice transplanted with cells (d 0) and allogeneic BALB/C T cells (d 2). Starting on day 3, mice received 5 doses of RG-7112 or vehicle every other day. Spleen cells were harvested on day 12 post allogeneic HCT. The sorted cells were used to recall the immune survival experiments. Similar results were obtained in three independent experiments.

Fig. 17: umap shows CD45 ⁺ And donor-derived (H-2 kb) ⁺ )TCRβ ⁺ CD8 ⁺ Marker expression on T cells.

(a, b) Umap shows live CD45 at random selection ⁺ Cell (a) and donor-derived (H-2 kb) ⁺ )TCRβ ⁺ CD8 ⁺ Marker expression on T cells (b) from small BALB/c bearing leukemia that have undergone allogeneic HCT And (3) mice.

Fig. 18: MDM2 inhibition results in increased levels of CD127 and Bcl-2 in CD 8T cells.

(a-d) scatter plots and representative histograms show CD8 isolated from spleen on day 12 after allogeneic HCT in BALB/C mice bearing WEHI-3B leukemia, transplanted with C57BL/6BM plus allogeneic C57BL/6T cells and treated with vehicle or MDM2 inhibitor RG-7112 ⁺ Expression of CD127 (k, l), bcl-2 (m, n) in cells. Mean ± SEM of each group of n=14-19 biologically independent animals from 2 experiments are shown, and p-values are calculated using a double sided Mann-Whitney U test.

Fig. 19: gating strategies for identifying primary AML blasts and MDM2 inhibition in PBMCs increased p53 in primary AML patient blasts.

(a) Flow cytometry charts show a gating strategy to identify primary AML blast cells in patient-derived PBMCs. The gating cells were singlet, living cells (dead living cells identified as negative for dye) and the marker CD34 ⁺ Or CD117 (cKIT) ⁺ Positive (gating of CD 34-positive cells is shown here). The selection markers are expressed based on information markers on AML cells at the time of initial diagnosis.

(b) After 48 hours of in vitro treatment with RG-7112 (2 μm), the cumulative p53 level of primary AML patient blast cells was determined by flow cytometry and shown as MFI for n=23 biologically independent patients. The MFI of p53 from control treated cells was set to 1.0. P-values were calculated using a two-sided Wilcoxon paired symbol rank test and are shown in the figure.

(c, d) histograms (c) and (d) show the fold change in MFI of p53 expression on primary AML blasts of representative patients after 48 hours of treatment with MDM2 inhibitor RG-7112 at the concentrations indicated, which is the mean ± SEM from one experiment performed in triplicate. MFI from control treated cells was set to 1.0 and p-values were calculated using a double sided Student unpaired t-test.

Fig. 20: MDM2 inhibition results in up-regulation of TRAIL-R1/R2 protein in primary AML patient blast cells.

(a) After 48 hours of in vitro treatment with RG-7112 (2 μm), the cumulative TRAIL-R1 level of primary AML patient blast cells was determined by flow cytometry and shown as MFI for n=23 independent patients. The MFI of TRAIL-R1 from control treated cells was set to 1.0. P-values were calculated using a two-sided Wilcoxon paired symbol rank test and are shown in the figure.

(b, c) histogram (b) and graph (c) show the fold change in MFI of TRAIL-R1 expression on primary AML blasts of representative patients after 48 hours of treatment with the indicated concentrations of MDM2 inhibitor RG-7112, which is the mean ± SEM from one experiment performed in triplicate. MFI from control treated cells was set to 1.0 and p-values were calculated using a double sided Student unpaired t-test.

(d) After 48 hours of in vitro treatment with RG-7112 (2 μm), the cumulative TRAIL-R2 levels of primary AML patient blast cells were determined by flow cytometry and shown as MFI for n=23 biologically independent patients. The MFI of TRAIL-R1 from control treated cells was set to 1.0. P-values were calculated using a two-sided Wilcoxon paired symbol rank test and are shown in the figure.

(e) The histogram shows the fold change in MFI of TRAIL-R2 expression on primary AML blasts of representative patients after 48 hours of treatment with the indicated concentrations of MDM2 inhibitor RG-7112, which is the mean ± SEM from one experiment performed in triplicate. MFI from control treated cells was set to 1.0 and p-values were calculated using a double sided Student unpaired t-test.

Fig. 21: MDM2 inhibition results in upregulation of TRAIL-R1/R2 mRNA in primary AML blasts of patient # 56. Purity control of AML xenograft mouse model using primary AML blasts of patient #56

(a) The bar graph shows TRAIL-R1/R2 protein levels (MFI) when primary AML blasts of patient #56 were exposed to MDM2 inhibition (RG). Human leukemia cells (without prior MDM2 inhibition) were used for survival studies in xenograft experiments (as shown in figure 4).

(b) Representative flow cytometry plots indicated AML cell enrichment prior to transfer to immunodeficient mice. The gating cells were singlet, viable cells (dead viable cell identification dye (fixable viability dye) negative) and human CD45 ⁺ 。

Fig. 22: MDM2 inhibition results in upregulation of TRAIL-R1/R2 mRNA in primary AML blasts in patient # 57. Purity of AML cells prior to metastasis and survival studies.

(a) The bar graph shows TRAIL-R1/R2 protein levels (MFI) when primary AML blast cells of patient #57 are exposed to MDM2 inhibition (RG). Human leukemia cells (without prior MDM2 inhibition) were used for survival studies in xenograft experiments.

(b) Representative flow cytometry patterns indicated the transfer to immunodeficiency Rag2 ^–/– Il2rγ ^–/– Mice were previously enriched for AML cells. The gating cells were singlet, viable cells (dead viable cells identified as dye negative) and human CD45 ⁺ 。

(c) Shows Rag2 after transfer of primary human AML cells ^–/– Il2rγ ^–/– Percent survival of recipient mice (patient # 57). As shown, mice are injected with additional human T cells (peripheral blood isolated from healthy donors) and/or treated with vehicle or MDM2 inhibitor RG-7112. N=8 independent animals from three experiments are shown and p-values are calculated using a two-sided Mantel-Cox test.

Fig. 23: pre-transplant p53 ^-/- P53 knockout efficacy in OCI-AML3 cells.

(a) Representative flow cytometry plots indicate p53 knockout efficacy in OCI-AML3 cells prior to transplantation. Cells were cultured in 20% FCS RPMI medium containing 1. Mu.g/ml doxycycline and 50. Mu.g/ml blasticidin for a minimum of 7 days. The gated cells are singlet and viable (dead viable cells identify dye negative). Cells with stable knockout efficiency were shown to be GFP ⁺ RFP ⁺ A population. Similar results were obtained in two independent experiments.

Fig. 24: increasing the oncogenic mutations FIP1L 1-PDGFR-alpha and cKIT-D816V of MDM2 in bone marrow BM cells makes AML susceptible to MDM2 inhibitor/T cell effects.

(a) 33000 primary murine BM cells transduced with FLT3-ITD, KRAS-G12D, cKIT-D816V, JAK-V617F or FIP1L 1-PDGFR-alpha at transfer and 5 x 10 ⁶ Spleens of mice 26 days after the BALB/cBM cells.

(b) The bar graph shows the weights of the different groups of spleens shown in (a).

(c) All CD45 in mouse BM from (a) quantified by flow cytometry ⁺ Oncogene transduction of cells (GFP ⁺ ) Percentage of cells.

(d) MDM2 protein (MFI) in primary mouse BM cells transduced with FLT3-ITD, KRAS-G12D, cKIT-D816V, JAK-V617F, FIP1L 1-PDGFR-alpha, BCR-ABL or c-myc as shown.

(e) MDM4 protein (MFI) in primary BM cells transduced with FLT3-ITD, KRAS-G12D, cKIT-D816V, JAK-V617F, FIP1L 1-PDGFR-alpha, BCR-ABL or c-myc as shown.

(f) Western blot shows the amount of MDM2 and loading control (. Beta. -actin) in primary murine BM cells transduced with the indicated FLT3-ITD, KRAS-G12D, cKIT-D816V, JAK-V617F, FIP L1-PDGFR-alpha, BCR-ABL or c-myc.

(g) The bar graph shows the ratio of MDM2/β -actin in primary murine BM cells transduced with FLT3-ITD, KRAS-G12D, cKIT-D816V, JAK-V617F, FIP1L1-PDGFR- α, BCR-ABL or c-myc. The ratio is normalized to EV (null vector). Two experiments were performed using biological replicates (BM from different mice) and the data pooled.

(h) The percent survival of BALB/C recipient mice after transferring FIP1L 1-PDGFR-alpha-tg transduced BM cells (BALB/C background) and after 30 days of allogeneic C57BL/6BM is shown. Mice received allogeneic C57BL/6CD3 on day 2 post BM transfer ⁺ T cells and treated with vehicle or MDM2 inhibitors.

(i) The percent survival of BALB/C receptor mice after transfer of cKIT-D816V-tg transduced BM cells (BALB/C background) and 30 days after allogeneic C57BL/6BM is shown. Mice received allogeneic C57BL/6CD3 on day 2 post BM transfer ⁺ T cells and treated with vehicle or MDM2 inhibitors.

Fig. 25: MDM2 and MDMX inhibit up-regulated MHC class I and II molecules.

(a) Microarray-based analysis of HLA class I and class II expression levels in OCI-AML3 cells after 24 hours of treatment with DMSO, RG-7112 (1 μm) or HDM-201 (200 nM) showed tiling from robust multichip mean (RMA) signal values, n=6 biologically independent samples/group.

(b, C) this figure shows the fold change in MFI of HLA-C (b), HLA-DR (C) expression on wild type OCI-AML3 (p53+/+) or p53 knockout (p 53-/-) OCI-AML3 cells after 72 hours of in vitro treatment with the indicated concentrations of MDM2 inhibitor HDM201, which is the mean ± SEM from an independent experiment of n=4. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(d, e) the graph shows the fold change in MFI of HLA-C (d) and HLA-DR (e) expression on OCI-AML3 cells after 72 hours of treatment with the indicated concentrations of MDMX inhibitor XI-006, which is the mean ± SEM from an independent experiment with n=7. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

Fig. 26: MDM2 inhibition increases p53 and MHC class II expression in malignant WEHI-3B, but does not increase p53 and MHC class II expression in non-malignant 32D cells.

(a) Western blot shows MDM2, p53 and loading control (GAPDH) expression in WEHI-3B cells exposed to DMSO, RG-7112 (0.5. Mu.M, 1. Mu.M) or 1000ng/ml doxorubicin for 4 hours.

(b) The graph shows the fold change in MFI of MHC class II expression on WEHI-3B cells after 72 hours of treatment with the indicated concentration of MDM2 inhibitor RG-7112, which is the mean ± SEM from n=6 independent experiments. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(c) Representative flow cytometry histograms describe the Mean Fluorescence Intensity (MFI) of MHC class II expression on WEHI-3B cells after 72 hours of treatment with the indicated concentrations of MDM2 inhibitor RG-7112.

(d) Western blot shows expression of MDM2, p53 and loading control (GAPDH) in WEHI-3B cells exposed to DMSO, HDM201 (100 nm,200 nm), or 1000ng/ml doxorubicin for 4 hours.

(e) The graph shows the fold change in MFI of MHC class II expression on WEHI-3B cells after 72 hours of treatment with the indicated concentration of MDM2 inhibitor HDM201, which is the mean ± SEM from independent experiments of n=4-6. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(f) Representative flow cytometry histograms describe the Mean Fluorescence Intensity (MFI) of MHC class II expression on WEHI-3B cells after 72 hours of treatment with the indicated concentrations of the MDM2 inhibitor HDM 201.

(g) Western blot shows expression of MDM2, p53 and loading control (GAPDH) in W32D cells exposed to DMSO, HDM201 (100 nm,200 nm), or 1000ng/ml doxorubicin for 4 hours.

(h) The graph shows fold change in MFI of MHC class II expression on 32D cells after 72 hours of treatment with the indicated concentration of MDM2 inhibitor HDM201, which is the mean ± SEM of n=4-6 independent experiments. The MFI of the control treated cells was set to 1.0. P-values were calculated using a double sided Student unpaired t-test.

(i) Representative flow cytometry histograms describe the Mean Fluorescence Intensity (MFI) of MHC class II expression on 32D cells after 72 hours of treatment with the indicated concentrations of MDM2 inhibitor HDM 201.

Fig. 27: graphic summary

The simplified schematic shows the proposed mechanism of action induced immune sensitivity of MDM 2-induced AML cells to T cells. MDM2 inhibition increased p53 levels. p53 translocates to the nucleus where it activates transcription of MHC class I and II and TRAIL-R1/2. Increased MHC II expression results in T cell priming, thereby enhancing their longevity and activation, with continued cytokine production. TRAIL-R upregulation on AML cells increases their sensitivity to T-cell induced TRAIL-mediated apoptosis, leading to activation of TRAIL-R1/2 downstream pathways (caspase-8, caspase-3, PARP) in AML cells.

Examples

The invention is further illustrated by the following examples. These examples are not intended to limit the scope of the invention, but rather to represent preferred embodiments of aspects of the invention, which are provided to better illustrate the invention described herein.

Method employed in the examples

Isolation and culture of patient-derived Peripheral Blood Mononuclear Cells (PBMC)

Human sample collection and analysis was approved by the institutional ethical review board of the university of Freiburg, germany, medical center (protocol number 100/20). Written informed consent was obtained from each patient. All human data analysis was performed in accordance with relevant ethical regulations. The characteristics of the patient are shown in table 1.

Isolation of human Peripheral Blood Mononuclear Cells (PBMC)

Human peripheral blood was collected in sterile EDTA-coated S-Monovette (Sarstedt, germany). Blood was diluted 1:1 with PBS and spread on 1 volume of human Pancoll (PAN-Biotech, germany). Gradient centrifugation was performed at 300 Xg without braking (acceleration: 9, deceleration: 1) at room temperature for 30 minutes to isolate PBMC. The mesophase containing the isolated PBMCs was aspirated and washed three times with PBS; 1 time at 300 Xg and then 2 times at 200 Xg for 10 minutes.

⁺ Isolation of CD4T cells from human PBMC

PBMC isolation was performed as described above. Enrichment of CD4 Using MACS cell separation System (Order No.130-045-101Miltenyi Biotec,USA) ⁺ T cells. For positive selection, anti-human CD4 was used ⁺ Microbeads (Miltenyi Biotec, USA). CD4 as assessed by flow cytometry ⁺ T cell purity is at least 90%.

Primary healthy donor PBMC and primary AML blast cells

Primary cells were maintained in RPMI medium supplemented with 20% fetal bovine serum, 2mM L-glutamine and 100U/ml penicillin/streptomycin.

Exposure of primary AML blasts to MDM2 inhibition

PBMCs were isolated from the blood of AML patients by Ficoll gradient centrifugation according to the manufacturer's instructions (Sigma-Aldrich), seeded in 24-well plates at a density of 500,000 cells/well, and incubated for 48 hours in RPMI-medium (Invitrogen, germany) supplemented with 10% Fetal Calf Serum (FCS) with or without RG-7112 (Selleck Chemicals LlcUSA) or HDM-201 (Novartis, basel, switzerland), where the concentration of RG-7112 or HDM-201 was specified in each experiment.

T cell activation and cytotoxicity assays

After separation of donor blood by Ficoll gradient centrifugation, cytotoxic T cells for cytotoxicity assays were generated from peripheral blood T cells of healthy volunteer donors, enriched by negative selection (negative selection) using Pan T cell separation kit II (Miltenyi Biotech) and MACS cell separation system (Miltenyi Biotec) according to manufacturer's instructions. The obtained T cells were at least 90% pure as assessed by flow cytometry. Isolated CD3 ⁺ T cells were treated with 25. Mu.l Dynabeads on day 1 ^TM Human T activator CD3/CD28 (Gibco, thermo Fisher Scientific)/million T cells were stimulated and co-cultured for 7 days on day 2 after isolation with 30U/ml human interleukin-2 (IL-2) (PeproTech) stimulation.

Quantitative real-time PCR of human AML samples

Total RNA of isolated patient PBMC was isolated using the Qiagen Rneasy kit according to the manufacturer's instructions. PBMCs were seeded at a density of 1 million cells/well in 6-well plates, cultured in RPMI medium (Invitrogen) supplemented with 10% fetal bovine serum, and treated with RG-7112 (0.5 μm, 1 μm and 2 μm) for 12 hours. For cDNA synthesis, 1 μg of RNA was reverse transcribed using random hexamer primers (high capacity cDNA reverse transcription kit applied biosystems/ThermoFisher Scientific) and a multi-fragment reverse transcriptase (ThermoFisher Scientific). Quantitative RT-PCR was performed using SYBR Green gene expression master mix (Roche LightCycler 480SYBR Green I Master) and primers provided in Table 2. All reactions were performed in triplicate with 50ng cDNA, calibration and reproducibility measurements were performed in duplicate, relative expression was calculated using the Pfaffl Δct method, and all mRNA levels were normalized to the reference gene hGAPDH. Primer sequences are provided in table 2.

A mouse

C57BL/6 (H-2 Kb) and BALB/C (H-2 Kd) mice were purchased from Janvier Labs (France) or from the university of Freiburg medical center animal facilityLocal inventory of applications. Rag2 ^–/– Il2rγ ^–/– Mice were obtained from local inventory of the animal facilities at the university of frieburg medical center. Mice of 6-14 weeks of age were used, and only female or male donor/acceptor pairs were used. Animal protocols are defined by the animal ethics committeeFreiburg (Freiburg, germany) approval (scheme number: G17-093, G-20/96).

Graft Versus Leukemia (GVL) mouse model

GVL experiments (5) were performed as described above. Briefly, in use ¹³⁷ Following (sub) lethal irradiation of the Cs source, recipients are injected intravenously (i.v.) with leukemia cells +/-donor BM cells. Isolation of CD3 from donor spleen or peripheral blood of healthy donors ⁺ T cells were enriched by negative selection using Pan T cell isolation kit II (Miltenyi Biotech, USA) and MACS cell isolation system (Miltenyi Biotec) according to the manufacturer's instructions. The obtained T cells were at least 90% pure as assessed by flow cytometry. CD3 administration on day 2 post-BM implantation ⁺ T cells.

^{MLL-PTD FLT3-ITD} AML leukemia model

For AML ^{MLL-PTD FLT3-ITD} Leukemia model, intravenous engraftment of C57BL/6 receptor with 5,000 AML after lethal irradiation with 12Gy ^{MLL-PTD FLT3-ITD} Cells and 5 million BALB/cBM cells, two aliquots were taken 4 hours apart. As previously reported, a total of 300,000 BALB/c (allograft model) spleen CD3 were introduced intravenously on day 2 post primary transplantation ⁺ T cells (19, 20).

WEHI-3B leukemia model

For the WEHI-3B leukemia model, BALB/C receptors were engrafted intravenously with 10Gy lethal irradiation with 5,000 AML (WEHI-3B) cells and 5 million C57/BL6 BM cells, with two aliquots at 4 hour intervals. Intravenous introduction of a total of 200,000C 57/BL6 (allograft) spleens CD3 on day 2 post primary transplantation ⁺ T cell。

OCI-AML3 xenograft model

For OCI-AML3 xenograft models ⁴ Rag2 was transplanted with 200,000 OCI-AML3 (wild-type or TRAIL-R2 knockdown) or 1 million OCI-AML3 (wild-type or p53 deficient) cells ^–/– Il2rγ ^–/– Receptors, as shown intravenously after sublethal irradiation with 5 Gy. Total 500,000 human CD3 isolated from peripheral blood of healthy donor was introduced intravenously on day 2 after primary transplantation ⁺ T cells.

Primary human AML xenograft model

For primary human AML xenograft model (21), rag2 was used ^–/– Il2rγ ^–/– A receptor. Primary human AML cells were isolated by FICOLL density centrifugation and isolated from CD3 by magnetic separation ⁺ Cells were removed. 1000 ten thousand CD3 were irradiated after 5Gy sublethal ⁺ Intravenous transplantation of depleted primary human AML cells. Intravenous introduction of a total of 50,000 human CD3 isolated from peripheral blood of healthy donor on day 2 after primary transplantation ⁺ T cells.

Leukemia model based on oncogenic mutations introduced in BM:

to induce leukemia based on certain oncogenic mutations, BALB/c receptors were transplanted with 30,000 BALB/c-derived BM cells transduced with cKIT-D816V or FIP1L 1-PDGFR-alpha. To induce the GVL effect, mice were irradiated at 10Gy in two aliquots, 4 hours apart. Then i.v. intravenous injection of recipient mice with five million C57/BL6 BM cells; 200,000C 57/BL6 splenic T cells were introduced intravenously on day 2 after allogeneic BM transfer. Spleen-derived T cells were enriched by MACS to remove all cells except CD3 positive cells.

Drug treatment in mouse models

On days 3-11 post-implantation mice were treated with RG-7112 (100 mg/kg) or vehicle (corn oil plus 5% DMSO) every other day (5 doses) by oral gavage. Purified anti-mouse CD253 (TRAIL) antibody or isotype control antibody was injected intraperitoneally at a dose of 12.5 μg/g body weight at days 4 and 8 post-implantation, as indicated in the respective experiments.

T cell phenotyping in GvL mouse model

T cell phenotyping experiments were performed using the WEHI-3B leukemia model. FACS analysis of spleen was performed on day 12 after WEHI-3B intravenous injection.

Leukemia cell line

The following leukemia cell lines were used: AML (active matrix liquid crystal display) ^{MLL-PTD FLT3-ITD} (22) (murine), WEHI-3B (23) (murine), and OCI-AML3 (human). AML (active matrix liquid crystal display) ^{MLL-PTD FLT3-ITD} Leukemia cells were supplied by b.r. blazar doctor (university of minnesota). All cell lines used for in vivo experiments were identified in DSMZ or multiplexing, germany. All cell lines were tested repeatedly for mycoplasma contamination and the results were negative.

Knock-out of p53 in OCI-AML3 cells

P53 knockout cells have been previously described (24). The p53 shRNA (p 53.1224) was cloned into a retroviral vector that co-expressed the red fluorescent protein and was inducible by doxycycline (24). The transfected cells were cultured in 20% FCS RPMI medium containing 1. Mu.g/ml doxycycline and 50. Mu.g/ml blasticidin to obtain stable knockout efficiency. Knockout of p53 was confirmed by Western blotting.

Knockout of TRAILR1/R2 in OCI-AML3 cells

HEK293T packaging cells were cultured in DMEM medium (Invitrogen, germany) supplemented with 10% Fetal Calf Serum (FCS). Chloramphenicol resistant lentiviral vectors pGFP-C-shLenti human TRAIL-R1 targeting shRNA (clone ID: TL308741A 5'-TTCGTCTCTGAGCAGCAAATGGAAAGCCA-3' (SEQ ID NO: 13)), pGFP-C-shLenti human TRAIL-R2 targeting shRNA (clone ID: TL300915B 5'-AGAGACTTGCCAAGCAGAAGATTGAGGAC-3' (SEQ ID NO: 14)), and pGFP-C-shLenti non-silencing shRNA controls (clone ID: TR30021[ AM1]5'-GCACTACCAGAGCTAACTCAGATAGTACT-3' (SEQ ID NO: 15)) were purchased from OriGene (USA). Lentiviral particles were generated by transfecting HEK293T cells using Lipofectamine 2000. 300,000 OCI-AML3 cells were transduced with lentiviral particles in the presence of 4. Mu.g/. Mu.l Polybrene (Merckmillipore). Knockdown of TRAIL-R1 and TRAIL-R2 was confirmed by FACS analysis.

Generation of TRAIL-R2 knockout OCI-AML3 cells

The CRISPR-Cas9 system expressing gRNA, cas9 protein and puromycin resistance gene (PMID: 25075903) was delivered using a neon transfection system (Invitrogen). TRAIL-R2 gRNA design (5'-CGCGGCGACAACGAGCACAA-3' (SEQ ID NO: 16)) and cloning into the lentiCRISPR v2 vector (Addgene plasmid # 52961) was performed according to the Zhang lab protocol (PMID: 31114586) as previously described. For delivery of the lentiCRISPR v2-TRAIL-R2 plasmid, 200.000OCI-AML3 cells were resuspended in resuspension buffer R (neon transfection system, invitrogen) in the presence of 2 μg plasmid. Cells were electroporated using a Neon transfection system in a 10. Mu.l Neon tip (Neon tip) at 1350V,35ms in a single pulse and immediately transferred to antibiotic-free recovery medium. TRAIL-R2 negative cells were isolated by cell sorting (BD Aria Fusion) and verified by flow cytometry analysis.

Isolation of mouse spleen cells and PMA/ionomycin stimulation

Single cell suspensions were obtained by triturating the spleen with a 70mm cell filter. Erythrocytes were lysed with 1mL of 1X RBC lysis buffer (ThermoFisher) on ice for 2 min, and the samples were washed with PBS and centrifuged at 400g for 7 min. Cells were re-stimulated in 2ml RPMI supplemented with Golgi-Stop and Golgi-Plug (1:1000, BD), phorbol 12-myristate 13-acetate (50 ng/ml, applichem) and ionomycin (500 ng/ml, invitrogen) for 5 hours at 37 ℃.

Microarray analysis

Total RNA of OCI-AML3 cells was extracted 24 hours after treatment with MDM2 inhibitor RG-7112 (2. Mu.M) or HDM-201 (500 nM) using the miRNeasy Mini kit (Qiagen, netherlands) and DNase (Qiagen, germany) according to the manufacturer's instructions. RNA integrity was analyzed by capillary electrophoresis using a fully automated nucleic acid fragment analysis system (Advanced Analytical Technologies, inc. Ames, IA). The RNA samples were further processed with the Affymetrix GeneChip Pico kit and hybridized to the Affymetrix Clariom S array as described by the manufacturer (Affymetrix, USA). The array was normalized by the robust multichip averaging implemented in the R/Bioconductor oligo package. Using the R/Bioconductor package 'gap' 48, the gene set enrichment was calculated using the pathway from condustpathdb 49 as the gene set, with a significance cut-off p < 0.05.

Microarray analysis (26) was performed as described previously. Microarray data is stored in the database GEO database under GEO accession number GSE 158103.

Western blot

OCI-AML3 cells were cultured for 4 hours in the presence or absence of 1mg/ml doxorubicin (pharmacy at the university of Freiburg medical center) or 1. Mu.M RG-7112 (Selleck Chemicals Ltc), and total protein extract (27) was prepared as described previously. To detect caspase activation, OCI-AML3 cells were treated with 1 μm RG-7112 for 72 hours and co-cultured with activated T cells at a ratio of 10:1 effector to target (E: T) for 4 hours. In some experiments, T cells were incubated with neutralizing antibodies against TRAIL (10. Mu.g/ml, MAB375, R & D Systems) or mouse IgG1 (# 401408, bioLegend) for 1 hour prior to co-culture. OCI-AML3 cells were analyzed after T cells were removed using Pan T cell isolation kit II.

Primary murine bone marrow cells transduced with EV (empty vector), FLT3-ITD, KRAS-G12D, cKIT-D816V, JAK2-V617F, FIP1L1-PDGFR- α, BCR-ABL or c-myc were sorted using a BD FACSAria III cell sorter (BD Bioscience, germany) to obtain GFP expressing cells and analyzed.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology) supplemented with phosphatase inhibitor mixture 2 (Sigma-Aldrich) and protein concentration was determined using the Pierce BCA protein assay kit (Life Technologies). Using NuPAGE ^TM LDS sample buffer and NuPAGE ^TM Sample reducing agent (Invitrogen) cell lysates were prepared for SDS-PAGE. Supernatant samples from cell-free supernatants were prepared using sample buffers containing SDS and Dithiothreitol (DTT). Using anti-p 53 (# 2527,Cell Signaling Technology), MDM2 (# 86934,Cell Signalin)g Technology), caspase 3 (# 9662,Cell Signaling Technology). anti-GAPDH (# GAPDH-71.1, sigma-Aldrich) and anti- β -actin (# 4970,Cell Signaling Technology) were used as loading controls. As the secondary antibody, horseradish peroxidase (HRP) -linked anti-rabbit or anti-mouse IgG (# 7074, #7076,Cell Signaling Technology) was used. Blotting signals were detected using WesternBright Quantum or Sirius HRP substrate (Advansta), imaged using ChemoCam Imager 3.2.0 (Intas Science Imaging Instruments GmbH), and quantified using ImageJ (NIH) software.

Flow cytometry

All antibodies used for flow cytometry analysis are listed in table 3. To exclude DEAD cells, the DEAD cell staining kit (Molecular probes, USA) or LIVE/DEAD can be immobilized using LIVE/DEAD according to the manufacturer's instructions ^TM Aqueous dead cell staining kit (Thermo Scientific) and True stage FcX (BioLegend) can be immobilized. For all fluorochrome conjugated antibodies, the optimal concentration was determined using titration experiments. Cells were incubated with the corresponding antibodies diluted in FACS buffer for 20 min at 4 ℃ for surface antigen staining. The cells were then washed with FACS buffer according to the manufacturer's instructions. For the mouse Bcl-2 assay, cells were fixed with a pre-warmed 3.7% formalin and a FACS buffer, then incubated in 90% methanol for 30 min, then Bcl-2 antibodies were added. Intracellular cytokine staining was performed using BD Cytofix/Cytoperm kit (BD Biosciences, germany) or Foxp 3/transcription factor staining buffer set (ThermoFisher) according to the manufacturer's instructions. For intracellular cytokine staining of mouse IFN-gamma, cells were stimulated with a dilution of a cell stimulation mixture (eBioscience, germany) containing PMA and ionomycin for an additional 4 hours according to the manufacturer's instructions prior to staining. Data were acquired on a BD LSR Fortessa Flow cytometer (BD Biosciences, germany) and analyzed using Flow Jo software version 10.4 (Tree Star, USA). For high-dimensional analysis, data was obtained on a Cytek Aurora (Cytek Biosciences) and pre-treated using Flow Jo software version 10.4 (Tree Star, USA) for both singlet and dead cell depletion and CD45 positive cells And (5) selecting.

Algorithm-directed high-dimensional analysis of spectral flow cytometry data

High-dimensional analysis was performed in the R environment. Two-dimensional UMAP (unified manifold approximation and projection) is generated using the UMAP package, and FlowSOM-based meta-clustering is performed as described by Brumelman et al (25).

Killing test

OCI-AML3 target cells were cultured in RMPI medium supplemented with 20% fcs for 72 hours in the presence or absence of 1 μm RG-7112, labeled with 0.5mM cell tracer violet BV421 (Thermo Fisher Scientific, germany) according to manufacturer's instructions, and co-cultured with effector T cells in 96 well plates at effector to target ratios of 10:1, 5:1, 2:1, and 1:1 for 16 hours. Cytotoxicity of effector T cells was measured using Zombie NIR APC/Cy7 (Biolegend).

Recombinant hTRAIL ((TNFSF 10, apo-2L, CD 253))For killing, ligand was added for 24 hours, 0.5 μg/ml (1:1000) for optimal killing, and 0.25 μg/ml (1:2000) for limiting killing conditions on OCI-AML3 target cells. The viability of the cells was assessed by LIVE/DEADTM fixable aqueous dead cell staining kit (Thermo Scientific). Data were acquired on a BD LSR Fortessa Flow cytometer (BD Biosciences) and analyzed using Flow Jo software version 10.4 (Tree Star).

Chromatin immunoprecipitation (ChIP analysis)

OCI-AML3 cells were treated with 2. Mu.M Rg-7112 for 12 hours and crosslinked with 1% formaldehyde at room temperature for 10 minutes and formaldehyde was inactivated to a final concentration of 125mM by the addition of glycine. Cells were resuspended in lysis buffer (1%SDS,10mM EDTA,50mM Tris-Cl, pH 8.0, protease inhibitor cocktail) and sonicated in Biorupter for 15 minutes at high power using a 30 second on/off program. After centrifugation at 16,000g for 5 min, the supernatant was collected and diluted with dilution buffer (20 mM Tris-Cl, pH 8.0,2mM EDTA,150mM NaCl,1% Triton X)-100, protease inhibitor cocktail) was diluted 10-fold. The prepared chromatin extracts were incubated with mouse IgG (sc-2025, santa-Cruz Biotechnology) or anti-p 53 antibody (sc-126, santa-Cruz Biotechnology) overnight at 4 ℃. Immunocomplexes were collected on a rotator using Dynabeads protein G (Invitrogen) beads for 2 hours at 4℃and washed 5 times with wash buffer (20 mM Tris-Cl, pH 8.0,2mM EDTA,0.1% SDS,0.5% NP-40,0.5M NaCl, protease inhibitor cocktail) and 4 times with TE buffer (10 mM Tris-Cl, pH 8.0,1mM EDTA). The DNA was eluted in elution buffer (100 mM NaHCO at 65 ℃C ₃ 1% SDS) for 6 hours and purified using a QIAquick gel extraction kit. Enrichment of bound DNA was measured using quantitative PCR and performed in a LightCycler 480 instrument (Roche, switzerland) using a LightCycler 480SYBR Green I Master kit (Roche, switzerland). Primer sequences are provided in table 2.

ChIP-qPCR data for each primer pair was expressed as percent input by calculating the amount of each specific DNA fragment in immunoprecipitates relative to the amount of that fragment in the input DNA.

Tumor cell lines

Human leukemia cell lines OCI-AML3, MOLM-13, and murine leukemia cell line WEHI-3B and non-malignant 32D cells were purchased from ATCC (American type culture Collection, manassas, virginia, USA) and cultured in RPMI medium supplemented with 10% FCS, 2mM L-glutamine, and 100U/ml penicillin/streptomycin.

Recall that immune experiments (Recall Immunity experiment)

For GvL recall immunization experiments, from C57BL/6BMT receptors (5 million BALB/C BM and 5,000 AML on day 12 post-allogeneic HCT ^{MLL-PTD/FLT3-ITD} Spleen cells were harvested from cells (d 0), 300,000 allogeneic T cells (d 2)). Then donor H-2kb was performed ⁺ CD3 ⁺ CD8 ⁺ FACS sorting of T cells. As assessed by flow cytometry, the cell purity was at least 90%. At 5 million BALB/c Bm and 5,000 AML ^{MLL-PTD/FLT3-ITD} After cell injection (d 0), we engrafted 100,000 sorted cells intravenously on day 2Into the secondary receptor.

Mouse bone marrow NK cell depletion

To deplete NK cells, naive BALB/c BM was isolated and surface stained for CD3 and NK 1.1. Sorting by FACS followed by NK1.1 exclusion ⁺ CD3 ^- Cells, leading to NK cell depletion in BM.

⁺ Depletion of CD8T cells in mouse bone marrow

To deplete CD8 ⁺ T cells, staining the extracted BM for CD3 and CD8 surface markers. In this case, CD3 was excluded by FACS sorting ⁺ CD8 ⁺ Cells, producing depleted CD8 ⁺ BM of T cells.

GVHD histological scoring

GVHD scoring (28) was performed as described previously. Organs small intestine, large intestine and liver were isolated and tissue sections were H & E stained and evaluated by pathologists blinded to the treatment group.

Extracellular flux assay

Extracellular flux assays were performed on a Seahorse analyzer (Agilent) recommended by the manufacturer. Briefly, 200000T cells were seeded in a 2mM glutamine supplemented Seahorse XF basal medium in each well of a 96-well Seahorse XF cell culture microplate. The cell culture plates were then subjected to non-CO at 37 ℃ ₂ Incubate in incubator for 45 min. The sensor cartridge port is loaded with glucose, oligomycin and 2-deoxyglucose (2-DG). Glycolytic stress tests were performed by measuring basal extracellular acidification rate (ECAR), followed by sequential injections of glucose (final concentration 10 mM), oligomycin (final concentration 1. Mu.M) and 2-DG (final concentration 50 mM).

Transfection of primary mouse BM cells with common oncogenic mutations or gene fusions

To generate EV-tg, FLT3-ITD-tg, KRASG12V-tg, cKITD816V-tg, JAK2V617F-tg, FIP1L1-PDGFRα -tg, BCRabl-tg, cMYC-tg Bm cells, BALB/c mice were injected with 100mg/kg 5-fluorouracil (Medac GmbH) four days prior to bone marrow harvesting. Mouse bone marrow was harvested and pre-stimulated overnight with growth factors (10 ng/mL mIL-3, 10ng/mL mIL-6 and 14.3ng/mL mSCF) as described previously (5, 29). Cells were transduced by 3 rounds of spin infection (2400 rpm,90min,32 ℃) by adding 2mL of retroviral supernatant supplemented with growth factors and 4. Mu.g/mL of polybrene every 12 hours.

Mass spectrometry sample preparation

Enrichment of CD8 from the spleen of recipient mice on day 12 post allogeneic HCT ⁺ T cells. T cells were incubated at a cell density of 2,000,000 cells/ml for 90min at 37℃in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco), 4mM L-glutamine, 100I.U./ml penicillin, 100. Mu.g/ml streptomycin, 100U/ml human recombinant IL-2 and 55. Mu.M beta. -mercaptoethanol. Thereafter, the cells were washed with PBS and the medium was exchanged with glucose-free RPMI 1640 medium, supplemented as above with 10mM U- ¹³ C-glucose. By U- ¹³ C-glucose was labeled for 50 minutes. One million cells were collected per sample and isolated from the cell culture medium by centrifugation at 500g for 5 minutes. Cells were washed with 500 μl PBS at 4deg.C, followed by another centrifugation step at 500g for 5 min at 4deg.C. After complete removal of the supernatant, the metabolites were extracted by re-suspending the cell pellet in 50 μl of methanol-acetonitrile-water (50:30:20) buffer pre-chilled for 30 minutes on dry ice. The samples were briefly vortexed and stored at-80 ℃.

Liquid chromatography-mass spectrometry (LC-MS)

LC-MS was performed using Agilent 1290Infinity II UHPLC, consistent with Bruker Impact II QTOF-MS operating in negative ion mode. The scanning range is 20-1050Da. Quality calibration is performed at the beginning of each run. LC separation was performed on a Hilicon ihlic (P) classical column (100×2.1 mM,5 μm particles) using a solvent gradient of 95% buffer B (90:10 acetonitrile buffer A) to 20% buffer A (20 mM ammonium carbonate+5. Mu.M aqueous methylene phosphate). Fl flow rate was 150. Mu.L/min. Auto-injector temperature was 5℃and injection volume was 2. Mu.L. Data processing for targeted analysis of absolute abundance of metabolites was performed using TASQ software (Bruker). The peak area of each metabolite was determined by manual peak integration. Only further analysis Metabolite peaks detected in > 80% of samples. The missing value was calculated as 50% of the lowest value detected in the whole sample group for the metabolite. Statistical comparisons were performed using unpaired double sided Student t test. Heat maps were generated using an mmetaabaanalysis 5.0 (30) as follows: carrying out logarithmic transformation and automatic scaling on peak area values; metabolites were clustered using hierarchical chiral clustering and the Ward clustering method based on euclidean distance. As described above ¹³ Data processing of C-glucose tracers, including correction of natural isotope abundance (31, 32).

Statistical analysis

For the sample size in the murine GVL survival experiment, a power analysis (Power analysis) was performed. An effect amount (effect size) of at least 1.06 was detected by determining the sample amount of at least n=10 per group by an efficacy of 80% to achieve a statistical significance of 0.05. Differences in animal survival were analyzed by the Mantel Cox test (Kaplan-Meier survival curve). The experiments were performed in a non-blind manner. For statistical analysis, unpaired t-test (double sided) was used. Data are presented as mean and SEM. (error bars). When the P value is <0.01, the difference is considered significant.

Example form

Table 1 AML patient characteristics

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Abbreviations: pats = patient, f = female, m = male, sAML = secondary AML, MDS = myelodysplastic syndrome

TABLE 2 primer sequences

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TABLE 3 flow cytometry antibodies

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Results of the examples

MDM2 inhibition increases the susceptibility of mouse and human AML cells to allogeneic T cell mediated cytotoxicity (vulnerability)

To examine the hypothesis that MDM2 inhibition will be synergistic with an alloimmune response, we treated mice with Bone Marrow (BM) alone or allogeneic HCT in combination with T cells. In mice bearing myelomonocytic leukemia cells (WEHI-3B), the addition of T cells to allogeneic BM grafts improved survival(FIG. 1 a). Treatment of leukemia bearing mice with MDM2 inhibitors in the absence of donor T cells increased survival but did not result in long term protection (fig. 1 a). Only when T cells were combined with MDM2 inhibition, most mice>80%) is protected for a long period of time (fig. 1 a). In AML ^{MLL-PTD/FLT3-ITD} Comparable survival patterns were observed in the model (FIG. 1 b) and the humanized mouse model using OCI-AML3 cells (FIG. 1 c). The T cell/MDM 2 inhibitor combination did not increase acute GVHD severity compared to T cell/vehicle (fig. 5 a-c).

When OCI-AML3 cells were exposed to MDM2 inhibition, the in vitro cytotoxicity of allogeneic T cells was higher (fig. 1 d). Consistently, cleaved caspase 3 was highest when T cells were combined with MDM2 inhibition (fig. 1 e-f).

To understand the mechanism responsible for the observed in vivo synergy, we exposed OCI-AML3 cells to MDM2 inhibition. Unbiased gene expression analysis showed that leukemia cells inhibited MDM2 up-regulated TRAIL-R1 and TRAIL-R2 (FIG. 1 g). Consistently, TRAIL-R1/TRAIL-R2-protein and TRAIL-R1/TRAIL-R2-RNA were increased following MDM2 inhibition in human OCI-AML3 cells (FIGS. 1h-i, FIGS. 6 a-j), MDM2 inhibition in mouse WEHI-3B cells (RG 7112, HDM 201) (FIGS. 7 a-h), or MDM2 inhibition in OCI-AML cells (XI-006) (FIGS. 8 a-c). Both RG7112 and HDM201 inhibit p53 degradation by preventing HDM2 binding. We used the p53 knockout OCI-AML3 cells to test whether TRAIL-R1/2 expression increase after MDM2 inhibition was dependent on p53, and found that p53 in p53 knockout cells induced a decrease in doxorubicin induction (fig. 9 a), while MDM2 inhibition induced p53 in p53 wild type cells (fig. 9 b). TRAIL-R1/2 expression increased with MDM2 inhibition (RG 7112 or HDM 201) in cells with intact p53, but not in p53 knockout cells (fig. 1j-k, fig. 9 c-d). In agreement, TRAIL is at p53 ^-/- Less apoptosis was induced in AML cells (fig. 9 e). Chromatin immunoprecipitation showed that p53 binds to the TRAIL-R1/2-promoter (FIG. 1 l-m).

Increased TRAIL-R1/2 expression following MDM2 inhibition contributes to the GVL effect

To determine the extent to which TRAIL-R1/2 expression contributes to enhanced GVL effects on MDM2 inhibition in AML cells, we blocked with anti-TRAIL ligandAntibody-treated mice. This reduced the protective effect of allogeneic T cell/MDM 2 inhibition (fig. 2 a). Interestingly, TRAIL ligand-deficient T cells (Tnfsf 10 ^{tm1b(KOMP)Wtsi} Mbpmu cd) also reduced the protective effect of MDM2 inhibition (FIG. 2 b). In addition, in vitro blocking of TRAIL-R1/2 reduced cytotoxicity of allogeneic T cells against MDM 2-inhibited exposed leukemia cells (FIGS. 2 c-e). TRAIL-R2 CRISPR-Cas knockdown AML cells (FIGS. 10 a-c) were less sensitive to allogeneic T cell/MDM 2 inhibition (FIG. 2 f). In WT-AML cells other than TRAIL-R2 ^-/- Therapeutic synergy of TRAIL plus MDM2 inhibition was observed in AML cells (fig. 2 g). T cells isolated from MDM2 inhibitor-treated mice showed higher glycolytic activity as measured by extracellular flux assay (FIG. 2 h-i). Through U- ¹³ An increase in glucose incorporation into several glycolytic intermediates confirms an increase in glycolytic flux (fig. 2 j). In addition, nucleotides and their precursors, particularly the nucleotides of the pyrimidine biosynthetic pathway and their precursors, were enriched in T cells isolated from MDM2 inhibitor treated mice (fig. 11 a-c). Increased glycolytic flux and nucleotide biosynthesis indicate stronger T cell activation, corresponding to higher GVL activity (6).

MDM2 inhibition promotes cytotoxicity and longevity of donor T cells

In the allogeneic HCT recipients receiving MDM2 inhibitors, donor CD8 is compared to those receiving the vehicle alone ⁺ T cells showed higher expression of the antitumor cytotoxic markers perforin and CD107a, and IFN-. Gamma., TNF and CD69, while CD8 ⁺ T cells did not increase overall (FIGS. 3a-h, 12a, 13 a-b). In the blank mice, CD107a, TNF and CD69 increased after MDM2 inhibition (FIGS. 14 a-d). CD8 ⁺ Depletion of T cells but not NK cells (fig. 15 a-b) resulted in loss of protective MDM2 inhibition (fig. 3 i), indicating that the anti-leukemia effect was mediated by CD8 ⁺ T-cell mediated. To understand whether recall immunity was generated under MDM2 inhibitor treatment, we isolated donor-type CD8 from mice bearing leukemia treated with vehicle or MDM2 inhibitor ⁺ T cells (fig. 16 a). T cells derived from mice treated with MDM2 inhibitors bearing leukemia are in the carrying relayImproved control of leukemia was elicited in mice with primary leukemia (fig. 3 j), indicating an anti-leukemia recall response. Effector T cells lacking CD27 showed a high antigen recall response (12), and we observed lower frequency cd8+cd27+tim3+ donor T cells in MDM2 inhibitor treated recipients (fig. 3k-m, fig. 17). T cells in MDM2 inhibitor treated mice exhibited a life span characteristic including high Bcl-2 and IL-7R (CD 127) (13) (fig. 18 a-d).

MDM2 inhibition in primary human AML cells results in TRAIL-1/2 expression

To verify our findings in a mouse model in human cells, we studied the effect of MDM 2-inhibition on primary human AML cells. MDM2 inhibition increased the level of p53 (fig. 19 a-d), showing activity of the hit target. MDM2 inhibition also increased the levels of TRAIL-R1 and TRAIL-R2RNA (FIGS. 4 a-d) and protein (FIGS. 20 a-e). The combination of MDM2 induction and allogeneic T cells enhanced the elimination of primary human AML cells in immunodeficient mice (fig. 4 e). AML cells showed increased TRAIL-R1/2 expression after MDM2 inhibition (FIG. 21a, FIGS. 22 a-c). The synergistic effect is dependent on intact p53, since human p53 ^-/- AML cells were resistant to MDM2 inhibitor/allogeneic T cell combinations (fig. 4f, fig. 23 a). The MDM2 inhibitor/allogeneic T cell combination caused activation of TRAIL-R1/2 downstream pathways (caspase 8, caspase 3, PARP) in human AML cells (fig. 4 g).

Oncogenic mutations that activate MDM2 expression confer increased sensitivity to T cell/MDM 2 inhibitor combinations.

To identify AML subtypes that may be particularly sensitive to T cell/MDM 2 inhibitor combinations, we studied the effects of various common oncogenic mutations or gene fusions (FLT 3-ITD, KRAS-G12D, cKIT-D816V, JAK-V617F, FIP1L-PDGFR- α, BCR-ABL and c-myc) on MDM 2. Mice receiving homologous BM transduced with the indicated oncogenic vectors developed splenomegaly and were treated with GFP ⁺ BM infiltration of transgenic cells (fig. 24 a-c). cKIT-D816V and FIP 1L-PDGFR-alpha induced MDM2 and MDM4 (FIG. 24D-g). Interestingly, allo-post BMT allogeneic T cell/MDM 2 inhibitor combinations carry FIP 1L-PDGFR-alpha-mutant and cKIT-D816V-mutationAre highly potent in mice with AML (fig. 24 h-i).

MDM2 inhibition increases MHC class I/II expression on AML cells in a p 53-dependent manner

Since it was shown that down-regulation of MHC genes and loss of mismatched HLA leads to recurrence of AML following allogeneic HCT (2, 4), we tested whether MDM2 inhibition could up-regulate MHC molecules on AML cells, thereby enhancing their recognition by allogeneic T cells.

Analysis of gene expression showed that HLAI and II were up-regulated after MDM2 inhibition (fig. 25 a). At the protein level, MDM2 inhibition increased HLA-C and HLA-DR expression on leukemia cells (FIGS. 4h-k, 25 b-C). HLA-DR was selected because its down-regulation was associated with AML recurrence after allogeneic HCT (2). Consistent with p 53-dependent regulation, HLA-C and HLA-DR did not increase with MDM2 inhibition in p53 knockdown OCI-AML3 cells (FIGS. 4 l-m). As a method for increasing p53 activity, MDMX inhibition (XI-006) (14) also increased HLA-C and HLA-DR (FIG. 25d, e). MDM2 inhibition caused increased MHC-II expression in primary human AML cells (FIGS. 4 n-o) and AML cell lines, but did not cause increased MHC-II expression in non-malignant cells (FIGS. 26 a-l). These findings indicate that targeting MDM 2-induced p53 down-regulation enhances anti-leukemia immunity after allogeneic HCT via MHC-II and TRAIL-R1/2 up-regulation in mice and humans (fig. 27).

Discussion of the embodiments

AML recurrence is caused by immune escape mechanisms (9). Our recent work has shown that AML cells produce lactate as an immune escape mechanism, interfering with T cell metabolism and effector functions (6). The second mechanism leading to recurrence is the blockade of IL-15 production by FLT3-ITD oncogenic signaling, leading to reduced immunogenicity of AML (5). In this study, we tested a new concept of relapse treatment, combining allogeneic reactivity of donor T cells with pharmacological approaches that reverse TRAIL-R1/2 and MHC-II down-regulation.

We have found that MDM2 inhibits the induction of TRAIL-R1/2 expression in primary human AML cells and AML cell lines. After TRAIL ligation, TRAIL death receptors assemble within their intracellular death domain a death-inducing signaling complex (DISC) consisting of FAS-related protein with death domain (FADD) and procaspase 8/10 (15). TRAIL-R activation was shown to have anti-tumor activity (16). Furthermore, MDM2 inhibition also increases MHC-II expression in primary human AML cells, which may provide a pharmacological point of intervention to reverse the MHC-II reduction observed in human AML recurrence following allogeneic HCT (2, 3).

Our observations are highly relevant clinically, as leukemic recurrence results in death of 57% of patients receiving allogeneic HCT (1, 17). We also delineate the immunological mechanism behind this observation, providing a scientific basis for AML relapse treatment with MDM2 inhibition and T cells, which would lead to phase I/II clinical trials.

Reference to the literature

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17.Nasilowska-Adamska,B.,et al.Mild chronic graft-versus-host disease may alleviate poor prognosis associated with FLT3 internal tandem duplication for adult acute myeloid leukemia following allogeneic stem cell transplantation with myeloablative conditioning in first complete remission:a retrospective study.Eur J Haematol.96,236-244(2016).

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23.Warner,N.L.,et al.A transplantable myelomonocytic leukemia in BALB-c mice:cytology,karyotype,and muramidase content.Journal of the National Cancer Institute 43,963-982(1969).24.Bric,A.,et al.Functional identification of tumor-suppressor genes through an in vivo RNA interference screen in a mouse lymphoma model.Cancer Cell 16,324-335(2009).

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inflammasome activation.Nat Commun.11,1659(2020).

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Sequence listing

<110> Albert-ludwig-Freiburg university (Albert-Ludwigs-university ä t Freiburg)

North Co., ltd (Novartis AG)

<120> MDM2 inhibitors for treating or preventing recurrence of hematological tumor after hematopoietic cell transplantation

<130> 2307/20WO

<150> EP 21184448.5

<151> 2021-07-08

<150> EP20197230.4

<151> 2020-09-21

<160> 16

<170> BiSSAP 1.3.6

<210> 1

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> Forward primer hTrailR1

<400> 1

gtgtgggtta caccaatgct tc 22

<210> 2

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer hTrailR1

<400> 2

cctggtttgc actgacatgc tg 22

<210> 3

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Forward primer hTrailR2

<400> 3

acagttgcag ccgtagtctt g 21

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer hTrailR2

<400> 4

ccaggtcgtt gtgagcttct 20

<210> 5

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Forward primer CDKN1A

<400> 5

gtggctctga ttggctttct g 21

<210> 6

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer CDKN1A

<400> 6

ctgaaaacag gcagcccaag 20

<210> 7

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Forward primer TNFRSF10A

<400> 7

ttcgcattcg gagttcaggg 20

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer TNFRSF10A

<400> 8

aagtggcaaa acgactccga 20

<210> 9

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> Forward primer TNFRSF10B

<400> 9

acgactggtg cgtcttgc 18

<210> 10

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer TNFRSF10B

<400> 10

aagacccttg tgctcgttgt c 21

<210> 11

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> Forward primer GAPDH

<400> 11

gtctcctctg acttcaacag cg 22

<210> 12

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer GAPDH

<400> 12

accaccctgt tgctgtagcc aa 22

<210> 13

<211> 29

<212> RNA

<213> artificial sequence

<220>

<223> TRAIL-R1 targeting shRNA

<400> 13

ttcgtctctg agcagcaaat ggaaagcca 29

<210> 14

<211> 29

<212> RNA

<213> artificial sequence

<220>

<223> TRAIL-R2 targeting shRNA

<400> 14

agagacttgc caagcagaag attgaggac 29

<210> 15

<211> 29

<212> RNA

<213> artificial sequence

<220>

<223> non-silencing shRNA control

<400> 15

gcactaccag agctaactca gatagtact 29

<210> 16

<211> 20

<212> RNA

<213> artificial sequence

<220>

<223> Trail-R2 gRNA design

<400> 16

cgcggcgaca acgagcacaa 20

Claims (19)

1. A mouse double minute 2 (MDM 2) inhibitor for use in the treatment and/or prevention of hematological tumor recurrence following Hematopoietic Cell Transplantation (HCT) in a patient.

2. The MDM2 inhibitor for use according to claim 1, wherein said hematological neoplasm is selected from the group comprising leukemia, lymphoma and myelodysplastic syndrome.

3. The MDM2 inhibitor for use according to any one of the preceding claims, wherein said hematological tumor is leukemia, preferably Acute Myelogenous Leukemia (AML).

4. The MDM2 inhibitor for use according to any one of the preceding claims, wherein said HCT is allogeneic HCT.

5. The MDM2 inhibitor for use according to any one of the preceding claims, wherein said HCT comprises a T cell.

6. The MDM2 inhibitor for use according to any one of the preceding claims, wherein said inhibitor is administered to the patient after HCT and before recurrence occurs.

7. The MDM2 inhibitor for use according to any one of claims 1-5, wherein said inhibitor is administered to a leukemia patient after occurrence of a post-HCT recurrence.

8. The MDM2 inhibitor for use according to any one of the preceding claims, wherein said inhibitor is selected from the group comprising RG7112 (RO 5045337), idanealin (RG 7388), AMG-232 (KRT-232), APG-115, BI-907828, CGM097, cisrimide (HDM-201) and Mi Lade maytansine (DS-3032 b) and pharmaceutically acceptable salts thereof.

9. The MDM2 inhibitor for use according to claim 8, wherein said inhibitor is cisco-line (HDM-201) or a pharmaceutically acceptable salt thereof.

10. The MDM2 inhibitor for use according to any one of the preceding claims, wherein administration of said MDM2 inhibitor results in up-regulation of one or more of TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1), TRAIL-R2, human Leukocyte Antigen (HLA) class I molecules and HLA class II molecules.

11. The MDM2 inhibitor for use according to any one of the preceding claims, wherein said treatment further comprises administering an allogeneic T cell transplant with and/or after HCT.

12. The MDM2 inhibitor for use according to claim 11, wherein said allogeneic T cell transplantation is donor lymphocyte infusion comprising lymphocytes but not hematopoietic stem cells.

13. The MDM2 inhibitor according to claim 11 or claim 12, wherein said donor of allogeneic T cell transplantation is also a donor of said HCT.

14. The MDM2 inhibitor according to any one of claims 11-13, wherein said MDM2 inhibitor is administered after said HCT and before and/or on the same day and/or after administration of said allogeneic T cell transplantation.

15. The MDM2 inhibitor for use according to any one of the preceding claims, wherein administration of said MDM2 inhibitor increases the cytotoxicity of cd8+ allogeneic T cells to cancer cells, wherein preferably the cytotoxicity of cd8+ allogeneic T cells is at least partially dependent on the interaction of TRAIL-R of said cancer cells and TRAIL-ligand (TRAIL-L) of cd8+ allogeneic T cells.

16. The MDM2 inhibitor for use according to any one of the preceding claims, wherein administration of said MDM2 inhibitor increases a graft versus leukemia or a graft versus lymphoma response, preferably wherein said graft versus leukemia response or said graft versus lymphoma response is mediated by cd8+ allogeneic T cells.

17. The MDM2 inhibitor for use according to any one of the preceding claims, wherein administration of said MDM2 inhibitor increases the expression of one or more of perforin, CD107a, IFN- γ, TNF and CD69 by cd8+ allogeneic T cells.

18. The MDM2 inhibitor for use according to any one of the preceding claims, wherein said treatment further comprises administration of an exporter 1 (XPO-1) inhibitor.

19. An XPO-1 inhibitor for use in the treatment and/or prophylaxis of a hematological tumor in a patient, wherein the treatment further comprises administration of a hematopoietic cell graft and an MDM2 inhibitor.