WO2025199203A1 - Compositions and methods for treating tp53-mutated myeloid neoplasms - Google Patents

Abstract

The present disclosure provides methods for assessing and/or treating subjects having CCUS, MN, or AML. In some embodiments, treatment of a subject with TTKi prevents progression of TP53^mut CCUS to TP53^mut MN. In other embodiments, methods are provided to treat TP53^mut MN with a TTKi, which functions by increasing genomic stability, increasing dysfunction of the spindle assembly checkpoint, and decreasing survival of aneuploidy cells. In another embodiment, the subject is treated with a TTKi in combination with at least one additional therapeutic agent, which may include a BCL-2 inhibitor, intensive chemotherapy, and a stem cell transplant.

Description

COMPOSITIONS AND METHODS FOR TREATING TP53-MUTATED MYELOID NEOPLASMS

BACKGROUND

[0001] TP53^mut MN represent 10-12% of all and 35-40% of therapy-related myeloid neoplasms (t-MN). Despite improvements in understanding of the mechanisms behind, and therapeutics for, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) as well as TP53 biology, the outcomes of TP53^mut MN remain extremely poor.²’ ^{3 20} Commonly used treatments including hypomethylating agents (HMA) or intensive chemotherapy led to little improvement in survival. While selective targeting of BCL-2 with the BH3-mimetic venetoclax has been revolutionary for AML, it was not associated with meaningful survival in TP53^mut MN.^{6 21} Further development of novel agents aimed at TP53^mut MN such as eprenetapopt (APR-246)²² and anti-CD47 antibody magrolimab²³ have been discontinued due to lack of efficacy. Thus, effective therapies for TP53^mut MN are urgently needed.

[0002] TP53^mut AML is more common in elderly (>60 years) population and its incidence increases from 4%, 16%, 19%, 17%, to 50% in those age <60, 61 -70, 71 -80, 81 -90, and >90, respectively.²⁴ With overall success of cancer-directed therapies, long-term complications of DNA-damaging therapies, including the development of TPSS ^ t-MN have come into focus.²⁵ Previously, it was hoped that the use of novel, non-cytotoxic approaches would result in decline in the incidence of t-MN and TPSSP^ MN. However, with expanding use and longer follow up, poly-(ADP-ribose) polymerase inhibitors (PARPi) are linked to t- MN.^26-28 Similarly, MN developing after radioneuclides²⁹ and chimeric-antigen receptor (CAR) T-cell therapies^30-34 have also been described (Gurney et al. JAMA One, in press). Thus, as the general population ages and the prevalence of cancer survivors rises, the incidence of TPS^ MN is expected to rise.

[0003] Clonal hematopoiesis (CH) and CCUS are recognized as the precursor to MN.^{5 35} For example, TP53^mut clone that evolves to MN can usually be identified years before the MN diagnosis.^36-40 On the other hand, whether TP53^mut clone imparts increased risk of a subsequent MN is a matter of active debate^{9 41} and emergence, disappearance, stability, and leukemic progression of the TP53^mut clone over time has been observed.¹²’ ³²’ ^{39 42-44} Median time from cytotoxic exposure to the development of TP53^mut MN is >5 years. Emerging evidence suggests that the latency may be shorter following the use of novel agents including CAR-T therapy^30-34 and PARPi,^26-28 suggesting that both hematopoietic stem cells (HSC) -intrinsic and -extrinsic factors contribute to the development of MN.

[0004] However, the ability to predict the behavior of the pre-malignant clone is limited. With the ever-expanding use sequencing technology, more cytopenic patients will be identified who harbor TP53^mut, but do not yet meet the criteria for a MN. Recognition of TP53^mut CH/CCUS may lead to overutilization of diagnostic testing, hesitancy to undergo lifesaving therapies, and induce anxiety. Therefore, informed guidelines on counseling, risk stratification, and surveillance strategies as well as additional clarification on the mechanisms of progression to TP53^mut MN is urgently needed.

[0005] Widespread genomic instability is one of the strongest predictors of inferior survival in both MDS and AML.⁵’ ²⁰’ ^{35 50} Genomic instability, manifesting as complex karyotype (CK), is relatively uncommon in MN (10-12%) but is highly prevalent in TP53^mut MN (70-80%).²’ ⁵¹ Enrichment of TP53^mut increases with increasing clonal complexity: from <5% in cases with diploid cytogenetics to >95% in those with >7 karyotypic abnormalities.⁴ Even within the high risk TP53^mut MN, patients with CK have shorter survival.^{2 4} On the other end of the spectrum — in CH and CCUS — cytogenetic abnormalities portends higher risk of MN progression and death.⁹’ ^{52 53}

[0006] TP53-deficient state facilitates two complementary processes — dysfunction of the spindle assembly checkpoint (SAC) predisposing to genomic instability, and survival of these aneuploid cells.^{17 54} Consequently, alterations of the SAC components are frequently observed in cancer.^{15 55} Since the SAC functions downstream to TP53, it is thought that SAC inhibition (SACi) can exert anti-cancer activity⁵⁶ and resensitize to the conventional chemotherapies even in TP53-deficient cells.⁵⁴ Threonine tyrosine kinase (TTK) is the central component of the SAC that ‘senses’ the lack of end-on attached microtubules to kinetochores, rapidly localizing other SAC components to the unattached kinetochores, and assisting the assembly of the mitotic checkpoint complex (MCC), ultimately preventing the progression to mitosis.⁵⁷ As expected, TTK is overexpressed in many malignancies and TCGA analyses confirmed TTK to be one of the most upregulated genes in TP53^mut cancers across the spectrum of cancers.^{58 59}

[0007] Therefore, TTK has emerged as an attractive therapeutic target.^13-18’ ⁵⁵’ ⁵⁶’ ^60-63 Although the exact mechanism is unclear, it is thought that TTK inhibition (TTKi) allows aneuploid cells to continue to divide, resulting in progressive genomic instability that jeopardizes their survival. Thus, targeting TTK in aneuploid TP53-deficient cells represent synthetic lethality.¹³’ ¹⁵

[0008] Both TP53^mut and CK are the most common causes of resistance to venetoclax based therapies.⁶⁴ Serial analysis of venetoclax naive and resistance cases showed acquisition of aneuploidy at the time of venetoclax resistance (VEN-r).²¹ In the absence of viable alternatives, venetoclax-based regimens have become de facto standard-of-care for TP53^mut AML and is being increasingly investigated in MDS. Therefore, identifying strategies to mitigate and reverse VEN-r is a critical means to improve survival in this subset and represents an urgent need of the day.

SUMMARY

[0009] The present disclosure provides materials and methods for assessing and/or treating patients with leukemia. In one embodiment, the disclosure provides a method of treating a subject diagnosed with TP53^mut myeloid neoplasms (TP53^mut MN), said method comprising administering a threonine tyrosine kinase inhibitor (TTKi) to the subject.

[0010] In another embodiment, a method is provided comprising treating TP53^mut MN in a subject, said method comprising the steps of (a) diagnosing the subject with TP53^mut MN, and (b) administering a TTKi to the subject. In still another embodiment, the present disclosure provides a method of increasing genomic stability in a subject, said method comprising the steps of (a) diagnosing the subject with TP53^mut MN, and (b) administering a TTKi to the subject. In yet another embodiment, a method is provided to increase dysfunction of the spindle assembly checkpoint (SAC) in a subject, said method comprising the steps of (a) diagnosing the subject with TP53^mut MN, and (b) administering a TTKi to the subject. In another embodiment, the present disclosure provides a method of decreasing survival of aneuploidy in cells in a subject, said method comprising the steps of (a) diagnosing the subject with TP53^mut MN, and (b) administering a TTKi to the subject.

[0011] In still another embodiment, the present disclosure provides a method of preventing progression of TP53^mut CCUS (Clonal Cytpoenia of Undetermined Significance) to TP53^mut MN in a subject, said method comprising administering a TTKi to the subject. Further provided, in another embodiment, is a method of restoring venetoclax sensitivity in venetoclax-resistant cells in a subject, said method comprising administering a TTKi to the subject.

[0012] In an aspect, the subject has a complex karyotype. In various aspects, the subject has 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomal abnormalities. In other aspects, the chromosomal abnormality is selected from the group consisting of a chromosomal deletion, duplication, inversion, and translocation. In still other aspects, the chromosomal abnormalities comprise chromosome 5 abnormalities, chromosome 7 abnormalities, or abnormalities in both chromosomes 5 and 7. In another aspect, the abnormalities are selected from deletions of chromosome 5q or 7q.

[0013] In an embodiment, the subject’s TP53^mut phenotype comprises one or more of T125_splice, H179, H179, R175, R213, G245, R248, R273, and R282 mutations in the TP53 gene. In another embodiment, the TP53^mut MN is selected from the group consisting of relapsed/refractory MN, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

[0014] In other embodiments, the TTKi is selected from the group consisting of CFI- 402257, S-81694 , BAY-1217389 , BOS-172722 , CC-671 , OSU-13 se, VRN-081569 , Empesertib , BAL-0891 , CCT-251455 , CFI-401870 , MPI-0479605 , and NTRC-0066-0. In one embodiment, the TTKi is CFI-402257.

[0015] In another embodiment, at least one additional therapeutic agent is administered to the subject in combination with the TTKi. In still another embodiment, the at least one additional therapeutic agent is selected from the group consisting of a BCL-2 inhibitor, intensive chemotherapy (doxorubicin, daunorubicin, idarubicin, cytarabine, azacitidine, decitabine), and a stem cell transplant. In yet another embodiment, the additional therapeutic agent is a BCL-2 inhibitor selected from the group consisting of venetoclax, navitoclax, obatoclax, and oblimersen sodium. In an aspect, the BCL-2 inhibitor is venetoclax.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A provides a multivariate analysis for survival in patients with TP53- mutated clonal cytopenia of undetermined significance (CCUS, n=40) and myelodysplastic syndrome with low blasts (n=173) show CK as an independent risk factor. Figure 1 B shows that isolated abnormalities of chromosome 5 are associated with lower variance allele frequency (VAF) of TP53^mut compared to those with additional abnormalities. Figure 1 C shows that isolated abnormalities of chromosome 7 are also associated with lower variance allele frequency (VAF) of TP53^mut compared to those with additional abnormalities. Figure 1 D shows that increasing clonal complexity is associated with higher TP53^mut VAF. For Figure 1 , Iso — isolated; Del — deletion; CK — complex karyotype; Non-rec. — non-recurrent.

[0017] Figure 2 describes overexpression of TTK mRNA in a wide variety of MN, including high-risk MN. Figure 2A shows that TTK was overexpressed in BBMC of MDS compared to healthy controls. Figure 2B shows that TKK was overexpressed in TP53^mut AML BMMC compared to TP53^wt AML. Figures 2C and D show that among AML cases, TTK overexpression was associated with increasing clonal complexity. Figure 2E shows that TKK was overexpressed in recurrent compared to primary AML. Figure 2F shows that TTK was overexpressed in TP53^mut myeloid cell lines compared to TP53^wt cell lines. For Figure 2, Mean ± SEM shown. * P<0.05; **P<0.01 ; *** P<0.005; ****P<0.001 . MDS - myelodysplastic syndrome; AML - acute myeloid leukemia; BMMC - bone marrow mononuclear cells.

[0018] Figure 3 demonstrates that addition of TKK inhibitor (TTKi) CFI-402257 at the GI25 dose resensitizes venetoclax-resistant U937 cells to venetoclax as measured by cell proliferation (Figure 3A) and induction of apoptosis (Figure 3B). For Figure 3, the experiments performed in triplicate, mean ± SEM shown. ****P<0.001. Ven - venetoclax.

[0019] Figure 4A demonstrates that TKK is overexpressed in TP53^mut myeloid cell line LI937 and MOLM-16 compared to TP53^wt OCI-AML3. Figure 4B provides results from myeloid cell lines LI937 and OCI-AML3 that were treated with increasing concentrations of TTK inhibitor (TTKi) CFI-402257 for up to 48 hours, and cell viability was measured using CCK-8 assay. Figure 4C demonstrates that TTKi reduces clonogenic potential of U937 cells. Figures 4D and 4E demonstrate that long-term treatment with TTKi at GI25 induces apoptosis in TP53^mut cell lines in the absence of appreciable polyploidy. In Figure 4, ****P<0.001 . Gl = growth inhibition; AS = Aneuploidy score as defined in Cohen-Sharir et al. Nature.¹⁶

[0020] Figure 5 shows that addition of TTK inhibitor (TTKi) CFI-402257 at the GI25 dose resensitizes venetoclax-resistant U937 cells to venetoclax as measured by (A) cell proliferation and (B) induction of apoptosis. For Figure 5, experiments were performed in triplicate, mean ± SEM shown. ****P<0.001 . Ven - venetoclax.

DETAILED DESCRIPTION

[0021] Disclosed herein are methods to effectively prevent and treat a highly aggressive leukemia that is expected to incur substantial morbidity and mortality in the near future. In some embodiments, the materials and methods provided herein can be used to diagnose a subject with a highly aggressive subset of leukemia, and treat the subject informed by their leukemia subset. Also disclosed herein is a strategy to diagnose an aggressive subset of leukemia at preneoplastic stages, or to diagnose an aggressive subset of leukemia at the myeloid neoplasm stage prior to progression to AML. In one embodiment, the disclosed method of treating TP53^mut MN in a subject comprises: (a) diagnosing the subject with TP53^mut MN; and (b) administering a threonine tyrosine kinase inhibitor (TTKi) to the subject.

[0022] Leukemia is the clonal proliferation of hematopoietic stem cells in the bone marrow. Leukemia is a common malignancy in children and adults that occurs when alterations in normal cell regulatory processes cause uncontrolled proliferation of hematopoietic stem cells in the bone marrow. Approximately one in 70 individuals develop leukemia in their lifetime. The invention generally provides methods of treatment of cancers of myeloid or lymphoid systems, including leukemias, lymphomas, and other cancers that typically are not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems. In one embodiment, the method comprises treating a subject diagnosed with TP53^mut myeloid neoplasm (TP53^mut MN), said method comprising administering a threonine tyrosine kinase inhibitor (TTKi) to the subject. As described herein, the long-term goal of the methods provided herein is to improve outcomes of TP53^mut MN. Proinflammatory cytokines secreted by the BM-MSC lead to the expansion of the preexisting TP53^mut clone via paracrine mechanisms. In one embodiment, loss of TP53 function may lead to TTK overexpression that in turn facilitates acquisition of aneuploidy and leukemic transformation. Inhibition of TTK results in robust antileukemic activity, both as a single agent or in combination with BCL-2 inhibitor venetoclax, representing a strategy to prevent and treat TP53^mut MN.

[0023] Leukemias can be generally classified into four broad subtypes: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myleloid leukemia (CML). ALL occurs more often in children, while the other subtypes are more common in adults (Davis et al., Am Fam Physician, 89(9): 731 -738, 2014).

[0024] ALL is a B-cell precursor (BCP) lineage (BCP-ALL) cancer or, less commonly, T- cell precursor lineage (T-ALL). Both comprise multiple subtypes commonly defined by structural chromosomal alterations that are initiating lesions, with secondary somatic (tumor- acquired) DNA copy number alterations and sequence mutations that contribute to leukemogenesis. Chromosomal alterations include aneuploidy and chromosomal rearrangements that result in oncogene deregulation or expression of chimeric fusion genes. KMT2A (MLL) rearrangements, particularly the t(4;11 )(q21 ;q23) translocation, are most frequent in infants (< 1 year of age) and are associated with poor outcome. High hyperdiploidy with gain of at least five chromosomes and ETV6-RUNX1 are each present in 25% to 30% of patients with childhood ALL but occur in less than 3% of young adults and are associated with favorable outcome. Conversely, BCR-ABL1 (Philadelphia [Ph] chromosome) -positive ALL composes 2% to 5% of childhood and 25% of adult ALL, and although historically associated with poor prognosis, outcomes have been markedly improved with the use of tyrosine kinase inhibitors (TKIs). The translocation t(1 ;19)(q23;p13) resulting in the TCF3-PBX1 fusion occurs in approximately 5% to 6% of childhood and adult BCP-ALLs. Secondary DNA deletions, gains, and mutations are characteristic of BCP-ALL, are important cooperating lesions in leukemogenesis, and may be acquired or enriched during disease progression. These include alterations of lymphoid transcription factors (IKZF1, PAX5, EBF1), cell-cycle regulation and tumor suppression (CDKN2A/CDKN2B, RB1), regulation of apoptosis, transcriptional regulation and coactivation ETV 6, ERG), and epigenetic alterations (lacobucci, J Clin Oncol, 35(9): 975-983, 2017).

[0025] AML is characterized by clonal expansion of immature blast cells in the peripheral blood and bone marrow, resulting in effective erythropoiesis and bone marrow failure. Early stages of pre-AML disease includes a condition referred to as clonal cytopenia of undetermined significance (CCUS), which can then progress to myeloid neoplasms (MN), which can further progress into AML. AML is a highly heterogeneous disease with a variable prognosis. It can result from genetic mutations, chromosomal translocations, or changes in molecular levels. About 97% of the embodiments have been studied to have genetic mutations. Despite its heterogeneity, it can be categorized into favorable, intermediate, or adverse-risk groups based on cytogenetics. The prognosis within these categories varies widely. The chromosomal translocations t (8;21 ), t (15;17), or inv (16) have a favorable prognosis with 3-year overall survival (OS) rate of about 66% and 33% in patients younger than 60 and older than 60 years of age respectively. People with t (9;11), monosomy 5 or 7, and normal cytogenetics (CN-AML) have an intermediate risk. A high risk of treatment failure and death was noted in people with t (6;9), inv (3). or 11q changes. The presence of c-KIT mutations in patients with t (8;21) increases the risk of relapse and decreases the OS. Frequently mutated genes in AML include NPM1, FLT3, RUNX1, DNMT3A, IDH1, IDH2, TET2, NRAS, CEBPa, WNT, and TP53. NPM1 mutations are associated with chemosensitivity in both young and old patients, as well as other genetic abnormalities including FLT3-ITD, FLT3-TKD, and IDH mutations. FLT3 '\s strongly expressed in hematopoietic stem cells, with important roles in cell survival and proliferation. Mutations involving the Internal tandem duplications (ITD) and the tyrosine kinase domain (TKD) of the FLT3 gene have been found in 20% of AML cases and 30% to 45% of CN-AML patients. Both the mutations activate FL T3 signaling, promoting blast proliferation. Patients with FLT3 mutations can have severe leukocytosis. FLT3-ITD mutations have been associated with an increased risk of relapse. Tyrosine kinase inhibitors (TKI) are being tested in FL T3 mutated AML patients. Unfortunately, when used alone, TKIs showed only a transient reduction of blasts, and even if initially effective, the subsequent acquisition of secondary mutations induces resistance over time. RUNX1 is an essential component of hematopoiesis; it is located on chromosome 21 and is frequently translocated with the ETO gene located on chromosome 8q22, creating an AML -ETO or t(8;21 )(q22;q22) AML which is seen in about 12% of AML cases. They are commonly associated with trisomy 13, trisomy 21 and show resistance to standard induction therapy (Vakiti et al., Acute Myeloid Leukemia, StatPearls, 2023).

[0026] CLL is characterized by the clonal proliferation and accumulation of mature, typically CD5-positive B-cells within the blood, bone marrow, lymph nodes, and spleen. The capacity to generate clonal B cells seems to be acquired at the hematopoietic stem cell (HSC) stage, suggesting that the primary leukemogenic event in CLL might involve multipotent, self-renewing HSCs (Hallek et al., Am J Hematol, 96: 1679-1705, 2021). Approximately 80% of all patients with CLL carry at least one of four common chromosomal alterations: a deletion in chromosome 13q14.3 (del[13q]), del(11q), del(17p), or trisomy 12.8 Del(13q) is the most common chromosomal alteration occurring in approx. 55% of all cases. An isolated del(13q14) is characterized by a benign course of the disease. The miRNAs miR-15a and 16-1 are located in the critical region of del(13q14)9 and regulate the expression of proteins that can inhibit apoptosis or that are involved in cell cycle progression. Deletions of the short arm of chromosome 17 (del(17p)) are found in 5%-8% of chemotherapy-naive patients. These deletions almost always include band 17p13, where the prominent tumor suppressor gene TP53 is located. Patients with CLL carrying a del(17p) clone show marked resistance against genotoxic chemotherapies. Mutations of TP53 are found in 4%-37% of patients with CLL, and have been associated with very poor prognosis in a number of studies (Zenz et al., Leukemia, 24: 2072-2079, 2010). Among cases with confirmed del(17p), the majority shows mutations in the remaining TP53 allele (>80%). In cases without del(17p), TP53 mutations are much rarer, but have a similarly detrimental effect on chemotherapy response and overall survival (OS). Deletions of the long arm of chromosome 11 (del(11q)) can be found in approx. 25% of chemotherapy-naive patients with advanced disease stages and 10% of patients with early-stage disease. These deletions frequently encompass band 11q23 harboring the gene ATM, which encodes for the proximal DNA damage response kinase ATM. In addition, patients carrying a del(11q) clone typically show a bulky lymphadenopathy, rapid progression, and reduced OS.16 Interestingly, some of the poor prognostic features of del(11q) were overcome by the use of chemoimmunotherapy.¹¹ Trisomy 12 is observed in 10%-20% of patients with CLL and is associated with an intermediate prognosis. The genes involved in the pathogenesis of CLL carrying a trisomy 12 are largely unknown.

[0027] CML is a myeloproliferative neoplasm caused by a reciprocal translocation [t(9;22)(q34;q 11 .2)] that leads to the fusion of ABL1 gene sequences (9q34) downstream of BCR gene sequences (22q11) and is cytogenetically visible as Philadelphia chromosome (Ph). BCR is a ubiquitously expressed cytoplasmic protein with multiple functionalities. ABL1 is also ubiquitously expressed and has several functions, including inhibition of cell cycle progression and proliferation, integrin signaling, and DNA repair. While ABL1 kinase activity is tightly controlled in physiological conditions, the chimeric BCR/ABL1 protein is a constitutively active tyrosine kinase that re-localizes to the cytoplasm. BCR/ABL1 activates numerous downstream pathways leading to increased proliferation, reduced apoptosis, abnormal adhesion and migration, and genetic instability. These activated signaling pathways collectively lead to malignant transformation. During the early (chronic) phase of CML (CP-CML), the myeloid cell compartment is expanded, but differentiation is maintained. Without effective therapy, CP-CML invariably progresses to blast phase (BP-CML), an acute leukemia of myeloid or lymphoid phenotype (Osman et al., Blood Rev, 49: 100825, 2021). [0028] Acute Leukemias and other blood cell malignancies that may be targeted include acute lymphoblastic, acute myeloid, acute lymphocytic, acute myelogenous leukemia, chronic myelogenous, hairy cell, erythroleukemia, lymphoblastic, myeloid, lymphocytic, myelogenous, leukemia, hairy cell, T-cell, monocytic, myeloblastic, granulocytic, gross, hand mirror-cell, basophilic, hemoblastic, histiocytic, leukopenic, lymphatic, Schilling's, stem cell, myelomonocytic, monocytic, prolymphocytic, promyelocytic, micromyeloblastic, megakaryoblastic, megakaryoctyic, Rieder cell, bovine, aleukemic, mast cell, myelocytic, plamsa cell, subleukemic, multiple myeloma, nonlymphocytic, chronic myelogenous leukemia, chronic lymphocytic leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, and myelodysplasia and chronic myelocytic leukemias.

[0029] Described herein is a method of preventing progression of TP53^mut CCUS to TP53^mut MN in a subject, said method comprising administering a TTKi to the subject. Preventing the progression of TP53^mut CCUS to TP53^mut MN is an atypical strategy, as the current status quo upon a CCUS diagnosis is non-intervention until the development of TP53^mut MN; at which point, no effective options are available. Virtually all TP53^mut MN patients have profound cytopenia, which is a major driver for morbidity and mortality. The paradigm of exploiting SAC dysfunction as a synthetic lethality as well as the strategy of long term and sustained TTK inhibition (TTKi) minimizing the adverse impact on normal hematopoiesis is particularly enticing.

[0030] Clonal cytopenia of undetermined significance (CCUS) is a preneoplastic disorder that often precedes TP53^mut MN and appears to be associated with favorable survival compared to TP53^mut MN.^8-10 CCUS can progress to MN once dysplasia, excess blasts, or MN-defining genetic lesions occur. Contrary to the prevalent perception, only 14% of TP53^mut CCUS progressed to MN, whereas the clone persisted without leukemic transformation in the remaining — raising the opportunity to diagnose or intervene early.¹¹ However, existing biomarkers cannot distinguish between the progressive and non-progressive TP53^mut CCUS. Therefore, devising strategies aimed at early identification and developing effective therapies are urgent unmet needs.

[0031] Myeloid neoplasms (MN) are a heterogenous clonal disorder of hematopietic stem cells characterized by cytomorphologic dysplasia, ineffective hematopoiesis, persistence of unexplained cytopenias, and morphological dysplasia. MN is further defined by a propensity to progress to bone marrow failure as well as risk of progression to AML. The disease category of “myeloid neoplasms with mutated TP53” encompasses MN, MN/AML, and AML. All of these subcategories possess TP53^mut, and are defined according to blast percentages. These diseases are grouped together because of their overall similar aggressive behavior irrespective of blast percentage, warranting a more unified treatment strategy across the blast spectrum. MN with TP53^mut is defined as having any kind of cytopenia, 0-9% bone marrow and blood blasts, and a multi-hit TP53^mut or TP53^mut (variant allele fraction (VAF)>10%) and complex karyotype (CK) often accompanied by loss of 17p. MN/AML with TP53^mut is defined as having any kind of cytopenia, 10-19% bone marrow or blood blasts, and any somatic TP53^mut (VAF>10%). AML with TP53^mutdoes not require cytopenia, and is defined by >20% bone marrow or blood blasts and any somatic TP53^mut (VAF>10%) (Arber et al., Blood, 140(11 ) :1200-1228, 2022).

[0032] The presence of multi-hit TP53 mutations in cytopenic MN corresponds to a highly aggressive disease with short survival. Unlike other MN, the prognosis of MN with multi-hit TP53 does not appear to depend on the blast percentage, although multi-hit TP53 abnormalities appear to be more common in cases with increased blasts (Weinberg et al., Blood Adv, 6(9): 2847-2853, 2022). Multi-hit TP53 can be confirmed by the presence of 2 or more distinct TP53 mutations (VAF > 10%) or a single TP53 mutation associated with (1 ) a cytogenetic deletion involving the TP53 locus at 17p13.1 ; (2) a VAF of >50%; or (3) copyneutral loss of heterozygosity (LOH) at the 17p TP53 locus (Bernard et al., Nat Med, 26(10): 1549-1556, 2020). In the absence of LOH information, the presence of a single TP53 mutation in the context of any complex karyotype is considered equivalent to a multi-hit TP53 (Grob et al., Blood, 139(15): 2347-2354, 2022). Complex karyotype alone in the absence of a TP53 mutation (even in the presence of 17p deletion) does not qualify for this category, as these cases have superior prognosis to TP53^mut MN (Haase et al., Leukemia, 33(7): 1747-1758, 2019).

[0033] As discussed above, progression to AML is defined by the clonal expansion of immature “blast cells” in the peripheral blood and bone marrow, resulting in ineffective erythropoiesis and bone marrow failure. Although the blast threshold of 20% defining AML remains, several additional genetic lesions are now considered to be defining of AML for myeloid neoplasms with >10% BM or blood blasts, which include ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2.

[0034] Leukemic transformation, from MN to AML, is the end result of complex interactions between bone marrow mesenchymal stromal cells (BM-MSC) and clonal hematopoietic stem cells (HSC). In HSC, the loss of functional TP53 leads to the dysregulation of the downstream spindle assembly checkpoint (SAC) facilitating chromosomal mis-segregation. A critical SAC component tyrosine threonine kinase (TTK or MPS-1 ) is overexpressed in a wide variety of cancers,^13-19 including TP53^mut and genomically unstable MN. Treatment with selective TTK inhibitors (TTKi) reduced proliferation and clonogenic potential while inducing apoptosis in TP53^mut MN cell lines and patient samples, suggesting TTKi as an attractive therapeutic strategy in this high-risk subset.

[0035] TP53^mut is associated with poor survival in nearly all cancers.^{91 92} Specifically, TP53 mutations in AML and MN are associated with a poor prognosis (Arber et al., Blood, 140: 1200-1228, 2022). Mutations in the TP53 genes are presents in 10-15% of AML cases and are enriched in poor-risk AML because of frequent additional cytogenetics or molecular alterations (Fleming et al., Blood, 142: 2029-2033, 2022). TP53^mut are seen in 10-12% of all MN and 35-40% of those with therapy-related myeloid neoplasms (t-MN). TP53^mut MN are highly chemoresistant cancers with median survival <1 year, and no existing or experimental therapies meaningfully improve survival in these patients.^2-7 Cytotoxic therapies, including stem cell transplants, used as a treatment of other cancers are the highest risk factors for the development of TP53^mut MN. The only potentially curative approach is allogeneic transplant resulting in long-term survival of 20-25% patients, though the risk of relapse remains very high (Ciurea et al., Blood, 131 (26): 2989-2992, 2018).

[0036] TP53 is located on chromosome 17p13 and is essential for cell cycle control and DNA damage response. Although the exact mechanism of leukemogenesis for mutant TP53 AML remains unknown, it has been shown that some TP53 mutations drive a dominant negative effect and typically occur in founding clones that expand after cytotoxic stress. Mutant TP53 is strongly associated with large structural and complex chromosomal aberrations, as illustrated by the co-occurrence of complex karyotypes (CK), which is associated with reduced overall survival in myeloid malignancies.

[0037] There are a wide spectrum of TP53 mutations in tumors, with their frequency varying greatly between different tumor types. TP53 is mutated in more than 90% of ovarian cancers, whereas less than 15% of AML have TP53^mut, suggesting that there may be some tissue-specific requirements for loss of wild type or gain of mutant TP53 functions (Pancancer analysis of whole genomes, Nature, 578(7793): 82-93, 2020). TP53mut has been reported to influence biological functions such as metastasis, sternness, epithelial to mesenchymal transition, amongst others (Aschauer et al., Biochem Soc Trans, 44: 460-466, 2016). There is strong enrichment for mutations in the DNA binding domain (DBD). DBD mutations are predominantly missense (-80%, as discussed above), including six “hotspot” codons (R175, R213, G245, R248, R273, and R282), which account for -25% of all TP53 mutations. Additional missense mutations include H179 and Y220 (Shah et al., Blood Cancer Journal, 13: 51 , 2023). In contrast, mutations that occur outside of the DBD are more likely to be nonsense or truncating mutations (-67%) than missense mutations (see Hainaut et al., Cold Spring Harb Perspect Med, 6(1 1 ): a026179, 2016). Mutant premature termination codons and frameshift mutations as well as most single amino-acid substitutions or deletions result in disruption of TP53 function. Loss of function (LOF) mutations in TP53 decrease expression of pro-apoptotic genes such as NOXA or PUMA, which are known transcriptional targets of TP53 (Aubrey et al., Cell Death Differ, 25: 104-1 13, 2018), and to a lesser degree increase the functional activation of the pro-apoptotic BAX and BAK proteins (Thijssen et al., Blood, 137: 2721 -2735, 2018). Specifically, the E180R mutant activates PUMA to sensitize an NRas^G12D;AML 1/ETO9a AML mouse model to chemotherapy (Klimovich et al., Oncogene, 41 : 1011 -1023, 2022). TP53 mutants V173M, I195S, R248Q, R273H, and insG282 represent a loss of TP53 wild type function, impacting the DNA damage response by repressing the transcriptional program in Ep-Myc lymphoma cell lines (Aubrey et al., Genes Dev, 32: 1420-1429, 2018). The R273H mutant represents a maintenance of TP53 wild type function, allowing TP53^mut to interact with SWI/SNF in MDA-468 cells to increase pro- angiogenic VEGFR2 expression (Pfister et al., Genes Dev, 29: 1298-1315). T125_splice is a splice site variant also found in TP53mut (Shah et al., Blood Cancer Journal, 13: 51 , 2023).

[0038] Beyond acquiring a TP53 mutation in one allele, most tumors lose the second allele by deletion or copy neutral loss of heterozygosity (Shirole et al., Elife, 5:e17929, 2016). TP53^mut tumors are reported to respond poorly to intensive chemotherapy, with OS rates of 6-9 months among subsets of AML. In a pooled analysis of phase II and II studies where patients were treated with BCL-2 inhibitor venetoclax and hypomethylating agent azacitidine, response rates were about 70% in patients with TP53^wt (with a median OS of 23.4 months) versus 41 % in patients with TP53^mut (with an OS of 5.2 months) (Daver et al., Am J Hematol, 98: 1176-1184, 2023).

[0039] Disclosed herein is a method of increasing genomic stability in a subject, said method comprising administering a TTKi to the subject. Widespread genomic instability is one of the strongest predictors of inferior survival in both MDS and AML. Genomic instability, manifesting as complex karyotype (CK), is relatively uncommon in MN (10-12%) but is highly prevalent in TP53^mut MN (70-80%). Genomic instability in AML is an important prognostic factor and is used for risk-stratification, with CK being associated with poor prognosis. TP53 mutations are highly present (60%) in AML patients with CK (Stengel et al., Leukemia, 31 : 705-71 1 , 2017). In older patients, (>60 years), only 10—44% of those with >3 cytogenetic abnormalities achieve complete remission (CR) after therapy, and for those with >5 chromosome abnormalities, the CR rates are significantly lower (7-26%) (Farag et al., Blood, 108: 63-73, 2006).

[0040] A key gene for maintenance of diploid karyotype in normal cells is TP53. TP53^wt has a role in suppressing genomic instability by inducing apoptosis in cells that exhibit a long pause at the mitotic checkpoint, which is indicative of DNA damage (Dalton et al., Oncogene, 29: 1929-1940, 2010). Mutation in TP53 arises before or after the first aneuploidy event, and these alterations may result in continued proliferation of aneuploidy cells or can trigger apoptosis (Fukasawa et al., Oncogene, 15: 1295-1302, 1997). These mutations are considered an early leukemogenic event in preleukemic stem cells. Enrichment of TP53^mut increases with increasing clonal complexity: from <5% in cases with diploid cytogenetics to >95% in those with >7 karyotypic abnormalities. Even within the high risk TP53^mut MN, patients with CK have shorter survival. During progression from MN to AML, TP53^mut clones often become bi-allelically mutated and genomically unstable, which is reflected by the strong association between bi-allelic TP53^mut and CK (Grob et al., Blood, 139(15): 2347- 2354, 2022). TP53-deficient state facilitates two complementary processes — dysfunction of the spindle assembly checkpoint (SAC) predisposing to genomic instability, and survival of these aneuploid cells. Consequently, alterations of the SAC components are frequently observed in cancer. For example, the most common chromosomal abnormality found in AML, t(8;21)(q22;q22), results in a fusion protein that impairs the spindle checkpoint and promotes aneuploidy (Boyapati, Blood, 109: 3963-3971 , 2003). Inactivation of the entire mitotic checkpoint can generate chromosomal mis-segregation leading to genomic instability or apoptosis in various cancers (Cahill et al., Nature, 392: 300-303, 1998). Since the SAC functions downstream to TP53, it is thought that SAC inhibition (SACi) can exert anti-cancer activity and resensitize to the conventional chemotherapies even in TP53-deficient cells.

[0041] In an embodiment, the method comprises reducing dysfunction of the spindle assembly checkpoint (SAC) in a subject, said method comprising administering a TTKi to the subject. Threonine tyrosine kinase (TTK) is a dual-specificity kinase that is critical for the recruitment of SAC proteins to unattached kinetochores, mitotic checkpoint complex (MCC) formation, and thus APC/C inhibition. TKK activation ultimately prevents cell cycle progression to mitosis. TTK is also required for chromosome alignment and error correction. Inhibition of TTK activity causes cells to prematurely exit mitosis with unattached chromosomes, resulting in severe chromosome mis-segregation, aneuploidy, and eventually cell death. While TTK expression is low in most organs, TTK is overexpressed in many malignancies and TCGA analyses confirmed TTK to be one of the most upregulated genes in TP53^mut cancers across the spectrum of cancers. Therefore, TTK has emerged as an attractive therapeutic target. Although the exact mechanism is unclear, it is thought that TTK inhibition (TTKi) allows aneuploid cells to continue to divide, resulting in progressive genomic instability that jeopardizes their survival. Thus, targeting TTK in aneuploid TP53-deficient cells represent synthetic lethality.

[0042] In one embodiment, the method comprises decreasing survival of aneuploidy cells in a subject, said method comprising administering a TTKi to the subject. Although genomic instability is defined as the increasing rate at which cells acquire new chromosomal alterations, aneuploidy is associated with the abnormal number of chromosomes in the karyotype at a specific point in time. Genomic instability, which manifests as a constant change in karyotype, is a hallmark of tumor malignancy and is thought to be one of the main causes of aneuploidy, the stable state of an imbalanced chromosome number (Torres et al., Cell, 143: 71-83, 2010). Aneuploidy and genomic instability have both been correlated with poor patient outcomes in multiple cancer types. The selective advantage of genomic instability to cancer growth is thought to derive from intra-tumor heterogeneity, facilitating the selection of chemotherapy resistant clones (Lee et al., Cancer Res, 71 : 1858-1870, 2011 ). In aneuploid cells, an abnormal chromosome count may deregulate cancer pathways or confer therapeutic resistance by duplication or loss of specific genes (Davoli et al., Cell, 155: 948- 962, 2013). Approaches that target genomic instability and aneuploidy in cancer include exploiting the cellular stress-state and resulting DNA damage caused by chromosomal segregation errors (Ohashi, Nat Common, 6: 7668, 2015). Another approach is to exploit the high activation of the SAC in many aneuploid and genomically unstable cells (Maia et al., Ann Oncol, 26: 2180-2192, 2015). If cancer cells are highly dependent on the SAC checkpoint due to their abnormal chromosome number, inhibition of SAC could selectively induce chromosome mis-segregation and cause cell death in the aneuploid or genomically unstable tumors.

[0043] In an embodiment, the method disclosed herein comprises administering a TTKi to a subject, wherein the subject has a complex karyotype. In another embodiment, the subject has a complex karyotype comprising 3, 4, 5, 6, 7, 8, 9, 10, or more chromosomal abnormalities. In other embodiments, the chromosomal abnormality is selected from the group consisting of a deletion, duplication, inversion, or translocation mutation.

[0044] TTK inhibitors serve to inhibit the SAC. Several TTK inhibitors have demonstrated efficacy in reducing xenograft growth in a variety of tumors in mice (Jemaa et al., Cell Death Differ, 20: 1532-1545, 2013; Kusakabe et al., J Med Chem, 58: 1760-1775, 2015), and can increase the efficacy of taxane chemotherapy in patient-derived xenograft mouse models (Wengner et al., Mol Cancer Ther, 15: 583-592, 2016). Definition of the patient population most likely to respond to a particular therapeutic is critical for the success of targeted therapies. However, determining the patient population that would benefit from TTK inhibition has been recognized as challenging for several reasons (Libouban et al., Oncotarget, 8(24): 38309-38325, 2017); (1) mutations in TTK are not detected at high frequencies in human cancers, and there is no relationship between mutated or activated TTK and malignancy status, (2) TTK is highly expressed in several cancer types, but the relationship between TTK expression and disease severity is complex and contradictive. For example, high TTK expression correlates with poor prognosis in hepatocellular carcinoma and HER2-positive breast cancer (Liu et al., Oncotarget, 6: 34309-34320, 2015), while low TTK expression correlates with poor patient outcomes in triple-negative breast cancer (Marie et al., PLoS One, 8: e63712, 2013). Disclosed herein is a methodology that identifies a specific patient population that would benefit from treatment with TTKi; patients diagnosed with TP53^mut MN and TP53^mut CCUS.

[0045] In another embodiment, the chromosomal abnormality can comprise chromosome 5 abnormalities, chromosome 7 abnormalities, or alterations in both chromosome 5 and 7. In an embodiment, the abnormalities are selected from deletion of chromosome 5q or 7q. Deletions or losses in chromosomes 5 or 7 are recurrent nonrandom abnormalities in AML and MN, and are associated with prior exposure to carcinogens or leukemogenic agents, with poor prognosis (Dabaja et al., Leukemia, 13: 869-872, 1999). With conventional intensive chemotherapy the outcome for patients with MN who have chromosome 5 and/or 7 abnormalities is as poor as that for patients with AML having chromosome 5 and/or 7 abnormalities (Fenaux et al., Semin Hematol, 33: 127-138, 1996). The only established therapy with curative potential for these poor-risk patients is allogenic stem cell transplantation (SCT) (Sierra, Blood, 100: 1997-2004, 2002), although this success is counterbalanced by a rather high transplant-related mortality as well as relapse (Gale et al., Bone Marrow Transplant, 16: 203-208, 1995). Furthermore, patients with chromosome 5 and 7 abnormalities have worse overall survival, relapse incidence, and transplant-related mortality than patients with either chromosome 5 or chromosome 7 abnormalities (van der Straaten et al., Haematologica, 90: 1339-1345, 2005).

[0046] To date, several types of small molecule compounds that inhibit TTK activity have been developed or identified. These small molecule compounds can be divided into four broad groups. The first group are compounds with N-phenylpyrimidin-2-amine scaffolds, which include Reversine, MPI-0479605, Mps1 -IN-3, AZ 3146, CC-671 , BOS-172722, Mps1- IN-2, NTRC 0066-0, and NMS-P715. The second group are compounds with N- phenylpyridine scaffolds, which include TC Mps112, Mps1-IN-1 , and CCT251455. The third group are compounds with 3-phenylindazole scaffolds, which include SP600125, CFI- 400936, and CFI-401870. The fourth group are compounds with five-membered bridged sixmembered heterocyclic scaffolds, which include Mps-BAY1 , Mps-BAY2a, Mps-BAY2b, BAY 1161909, BAY 1217389, CFI-402257, PF-7006, and PF-3837. TTK inhibitors have been tested in combination with microtubule-targeting agents to increase chromosomal separation errors and kill cancer cells more efficiently. For example, CC-671 (a highly selective inhibitor of TTK and CLK2) was found to inhibit the drug efflux activity of ABCG2 in lung cancer cells, thus increasing the level of intracellular chemotherapy and potentially improving the efficacy of chemotherapy in lung cancer (Wu et al., Cancer Science, 1 11 (8): 2872, 2020). Another group developed a TTK small molecule inhibitor, Mps1 -IN-3, which caused mitotic abnormalities in glioblastoma cells, and the combination of Mps1 -IN-3 with vincristine increased aneuploidy and cell death (Tannous et al., Journal of the National Cancer Institute, 105(17): 1322-1331 , 2013). Currently, five small molecule TTK inhibitors are in clinical trials: BAY-1217389 (NCT02366949), BAY-1 161909 (NCT02138812), BOS-172722 (NCT03329494), CFI-402257 (NCT02792465, NCT03568422), and S-81694

(NCT03411 161 ). All five inhibitors are being assessed in combination with paclitaxel, as this combination increased cancer sensitivity and mitigated side effects due to the reduced dosage used for the combination.

[0047] In one embodiment, the disclosed method comprises administering a TTKi to a subject in need thereof, wherein the TTKi is selected from a group consisting of CFI-402257, S-81694 (see WO2009156315A1 ), BAY-1217389 (see WO2014131738A2), BOS-172722 (see W02014037750A1 ), CC-671 (see WO2014113429A2), OSU-13 (see Wengner et al., Mol Cancer Ther, 15(4): 583-92, 2016), VRN-081569 (see Jung et al., Proceedings of the AACR Annual Meeting, Philadelphia (PA): AACR; Cancer Res 2021 ; 81 (13_Suppl): Abstract nr LB119), Empesertib (see WO2013087579A1 ), BAL-0891 (see Lane et al., Annals of Oncology, 33(S1 ): Abstract nr 42P, 2022), CCT-251455 (see WO2012123745A1 ), CFI- 401870 (see WO2013053051 A1 ), MPI-0479605 (see WO20101 11406A2), and NTRC-0066- 0 (see WO2015155042A1 ). Recently, an orally bioavailable, highly specific, and potent small molecule inhibitor of TTK (CFI-402257) has been described.^{95 96} Toxicology studies of CFI- 402257 in rodents and dogs showed no evidence of overt toxicity.⁹⁶ Importantly, CFI-402257 has shown promising activity against colon, ovarian, hepatocellular carcinoma, and breast cancer and is being studied in various solid malignancies (NCT05251714, NCT03568422, and NCT02792465).¹³’ ¹⁸’ ⁵⁶’ ⁶⁰’ ⁹⁷ TTK is overexpressed in high-risk MN including those with TP53^mut, CK, and in relapsed/ refractory MN. CFI-402257 readily and selectively induces apoptosis and reduced clonogenic potential in bone marrow mononuclear cells (BMMC) of TP53^mut AML compared to TP53^wt AML and healthy donors.

[0048] The “3+7” regimen (3 days of daunorubicin + 7 days of cytarabine), developed in the late 1970s, was the standard of care for nearly 40 years, producing estimated 5-year survivals of 30% to 35% in younger patients (age <60 years) and 10%-15% in older patients (age >60 years). While the clinical trials that led to the approval of the “3+7” regimen were conducted in a highly selective patient population, broad application of this intensive chemotherapy regimen was subsequently not well tolerated in older patients with multiple comorbidities (e.g., hypertension, diabetes, cardiac hypertension, ect...). Addition of other drugs and intensification of the cytarabine dose in the “3+7” regimen have failed to improve outcomes for patients (Preisler et al., Blood, 69(5): 1441 -1449, 1987). Current AML management relies largely on intensive chemotherapy and allogenic hematopoietic stem cell transplantation (HSCT) for younger patients who can tolerate intensive treatments (Dombret et al., Blood, 127(1 ): 53-61 , 2016). Intensive chemotherapy includes high dose cytarabine (HIDAC), which is now considered the standard of care for consolidation in younger patients with AML (Mayer et al., N Engl J Med, 331 : 896-903, 1994). In younger patients, complete remission (CR) rates of >80% may be reached, with 5-year overall survival (OS) -40%. In older patients, the use of hypomethylating agents has improved median and short-term OS but has not translated into improved cure rates, which remain disappointingly low.

[0049] Recent clinical innovation in AML treatment includes small molecule-targeted therapies, such as FLT3 and IDH inhibitors, and the BCL-2 inhibitor venetoclax. FLT3 mutations are molecular abnormalities present in 25-30% of AML patients. These abnormalities are associated with worse OS outcomes, regardless of cytogenetic risk. Midostaurin is an oral tyrosine kinase inhibitor that has demonstrated efficacy in inhibiting this abnormal gene. A reduction of peripheral circulating blasts was demonstrated in 7 of 20 patients with relapsed FLT3+ AML treated with single-agent Midostaurin (Kottaridis et al., Blood, 98(6): 1752-1759, 2001 ).

[0050] In an embodiment, the method disclosed herein comprises administering at least one additional therapeutic agent in combination with the TTKi. The additional therapeutic agent may consist of cydarabine, azacitidine, daunorubicin, decitabine, a FLT3 inhibitor, an IDH inhibitor, avapritinib, dasatinib, venetoclax, navitoclax, obatoclax, oblimersen sodium, oblimersen magrolimab (anti-CD47 monoclonal antibody), 6-mercaptopurine-methotrexate, ATRA, arsenic trioxide, idarubicin, gemtuzumab ozogamicin, fludarabine, filgrastim, and APR246. The additional therapeutic may also comprise a stem cell transplant.

[0051] Described herein is a method of restoring venetoclax sensitivity in venetoclax - resistant cells in a subject, said method comprising administering a TTKi to the subject. Intrinsic apoptosis relies on the balance between pro- and anti-apoptotic proteins inducing mitochondrial outer membrane permeabilization (MOMP), ultimately leading to caspasedependent cell death. In response to apoptotic stimuli, BAX and BAK form pores in the mitochondrial membrane, inducing mitochondrial outer membrane permeabilization (MOMP). This phenomenon is increased by the members of the BCL-2 protein family containing a single BH3 domain named “BH3-only proteins”, which have a pro-apoptotic role (BIM, NOXA, PUMA, BID). Conversely, MOMP is blocked by a series of proteins that have an anti- apoptotic role including BCL2, BCL-XL, and MCL1 . BCL2 is often overexpressed in AML cells, and is associated with poor prognosis and resistance to chemotherapy (Campos et al., Blood, 81 : 3091-3096, 1993). In another embodiment, the disclosed method comprises administering a BCL-2 inhibitor, wherein the BCL-2 inhibitor is selected from the group consisting of ABBV-453, ABBV-623, BCL-201 , LOXO-338, LP-108, TQB3909, ZN-d5, LP- 118, VOB560 (S65487), AZD0466, APG 2575, bcl-2 antisense oligodeoxynucleotide G3139, APG-1252, BGB-11417, GDC-0199, or venetoclax.

[0052] The rationale behind BCL-2 inhibitors is to induce intrinsic apoptosis by blocking anti-apoptotic proteins. The BCL-2 inhibitor venetoclax has undergone clinical trials in combination with hypomethylating agent azacitidine, and patients receiving the combination treatment exhibited better overall survival compared to patients that received azacitidine alone (DiNardo et al., Blood, 130(1 ): 2628, 2017). Mutations in particular genes can influence venetoclax efficacy in AML patients. CRISPR-Cas9 screens have consistently identified BAK, BAX, PUMA, and NOXA, as well as TP53 target genes as crucial regulators of venetoclast activity in vitro (Fischer, Oncogene, 36: 3943-3956, 2017).

EXAMPLES

[0053] Given the association of TP53^mut with poor survival in nearly all cancers^{91 92} including MN,^{5 35} it is commonly presumed that TP53^mut rnay also portend poor survival in CCUS. However, there is a lack of consensus if TP53^mut is independently associated with poor survival in CCUS. In a population study of 873 CCUS patients, TP53^mutwas highly predictive for subsequent MN (positive predictive value 0.82-0.9).⁴¹ In contrast, two population studies that included >400,000 participants each, did not find TP53^mut as an adverse-risk factor for subsequent leukemia.⁹’ ¹⁰

[0054] In a Mayo Clinic cohort of 173 CCUS patients (36 TP53^mut, 137 TP53^wt), survival was comparable in TP53^mut and TP53^wt CCUS.¹¹ While 5 (14%) TP53^mut CCUS progressed to MN (median time to progression: 13.5 months), the remainder tolerated the presence of the clone without progression. Survival of TP53^mut MDS was significantly shorter than TP53^mut CCUS as expected.

[0055] The presence of TP53^mut clones suggests high-risk of future MN, but additional cell-intrinsic and extrinsic factors are necessary to drive progression to myeloid neoplasm. Leukemic transformation is a clinically significant event as the survival of TP53^mut MN is dismal compared to the CCUS counterpart. Recognition of the collaborating alterations will lead to early diagnosis and opportunity to intervene, improving outcomes.

[0056] Example 1 : Characterization of the sequences of genomic and transcriptomic alterations that preclude leukemic transformation of TP53^mut CCUS.

[0057] In premalignant TP53mut HSC, loss of TP53 is followed by TTK overexpression and acquisition of cytogenetic abnormalities — resulting in leukemic progression. Using a combination of genomic and transcriptomic approaches, the development of aneuploidy in TP53-deficient cells as the leukemia-initiating event will be demonstrated. Differentially expressed TTK in TP53-deficient cells will be identified and validated. Finally, TTKi will lead to selective elimination of aneuploid cells, decreasing the risk of leukemic transformation.

[0058] It has been reported in a UK Biobank study that deletion 5q was the only copy number alteration (CNA) associated with a higher risk of both MDS and AML.¹⁰ In a large cohort of 213 cytopenic patients harboring TP53^mut (40 CCUS, 173 had myelodysplastic syndrome with <5% blasts) treated at Mayo Clinic/SA-MDS, cytogenetic abnormality was the only factor predicting shorter survival independently (Figure 1 A). In contrast, diagnostic dysplasia or biallelic TP53 loss were not independent predictors. Moreover, deletion of chromosomes 5q and/or 7q, but not the non-recurrent cytogenetic abnormalities, were associated with a larger TP53^mut clone size (Figure 1 B-D). These data follow a deterministic pattern, with abnormalities in chromosomes 5 and 7 being the early events in leukemic transformation. Deletion 5q appears to particularly serve as an early leukemia initiating event.

[0059] Taken together, the Figure 1 data suggests that the acquisition of aneuploidy may follow deterministic patterns with abnormalities of chromosomes 5 and 7 being the early events in leukemic transformation. Deletion 5q in particular appears to be an early leukemia initiating event.

[0060] Additional Approaches

[0061] Despite the broad consensus as to TP53^mut MN being associated with poor survival, there is an active debate as to how TP53^mut MN is defined.³’ ⁵’ ^{35 88}’ ¹⁰⁸ The discrepancies arise at least in part due to an inherent inability of the bulk sequencing techniques to accurately assign allelic status and establish ‘true’ loss of TP53 function.^{4 87}’ ¹⁰⁹ The current empiric variance allele frequency (VAF) cutoffs are neither rooted in biology nor an adequate surrogate for the allelic status, as demonstrated by a recent study.¹¹⁰ Moreover, the deterministic pattern of genomic changes immediately preceding leukemic transformation is not known, and while analysis of publicly available data strongly suggests TTK overexpression in MN, TTK expression in TP53^mut CCUS has not been characterized. Finally, the feasibility of targeting SAC downstream to TP53 with the aim of preventing the progression of TP53^mut CCUS to TP53^mut MN has not been attempted.

[0062] Proposed herein is a comprehensive and serial comparison of genetic, genomic, and transcriptomic alterations between progressive and non-progressive TP53^mut CCUS. Cell cycle and SAC genes will be serially quantified, including TTK before and after leukemic transformation. Finally, demonstration that genetic or pharmacological inhibition of TTK eliminates aneuploid cells will provide evidence to evaluate TTKi as a preventive strategy.

[0063] First, the genomic alterations associated with leukemic transformation in TP53^mut CClIS will be defined. The primary goal of this experiment is to determine if haploinsufficiency of chromosomes 5 and 7 are independently associated with an increased risk of progression to TP53^mut MN. Whole genome sequencing (WGS) profile of the enriched CD34+/CD38- HSCs will be performed for progressive and non-progressive TP53^mut CCUS (n=15 each) at up to 5 timepoints (at TP53^mut CCUS diagnosis, up to 3 interval samples, and at the last follow up for non-progressive TP53^mut CCUS and TP53^mut MN diagnosis for progressive TP53^mut CCUS). For WGS, 300 ng of genomic DNA will be used to create PCR- Free libraries using the standard DNA PCR-Free Prep Tagmentation Workflow (Illumina) to minimize coverage-bias. After quality control, libraries will be sequenced at an average coverage >1 OOx that is expected to identify nearly 100% of CNA that could be identified on the standard cytogenetic techniques.¹¹¹ Bioinformatic analyses will be performed in collaboration with Mayo Clinic and SAHMRI Data Science teams. Briefly, FASTQ files will be aligned to the hg38 reference genome using bwa-mem (v.0.7.10) and realignment will be performed using GATK (v4).¹¹²To identify single nucleotide variants (SNV), insertions/deletions (indels), and structural variants (SV) the workflow uses a combination of Mutect2,¹¹³ Strelka2,¹¹⁴ and Manta,¹¹⁵ respectively. Copy number alternations (CNA) will be detected using Pattern Copy Number Variation.¹¹⁶ Detection of chromothripsis will be based on a combination of SV and CNA data.¹¹⁷ Identified variants will be annotated using BioR framework¹¹⁸ using ClinVar, Human Gene Mutation Database, Mayo Biobank, and Exome Aggregation Consortium population frequencies. Primary readout from WSG experiment will be the characteristics TP53^mut, VAF, co-mutation pattern, and the association with CAN, including chromosome 5 and 7 CAN. Finally, the subclonal architecture of CCUS progressing to MN will be inferred by using ClonEvol¹¹⁹ and the clonal evolution across timepoints will be visualized by fish plot.

[0064] Next, the cell-of-origin for the leukemic transformation will be defined, and serial genomic changes before the leukemic transformation will be characterized. The primary goal of this experiment is to establish that colocalization of biallelic TP53^mut clone with chromosomal 5 and/or 7 abnormalities in the HSC and progenitor cells. Samples used in the prior experiment will undergo simultaneous single-cell DNA sequencing using the Tapestri Platform as described in detail,¹²⁰ except that the most promising CNA targets identified by WGS will be included in the customized panel. Briefly, bioinformatics analyses will be performed using Tapestri Pipeline to perform adapter trimming, sequence alignment, barcode correction, cell finding, and variant calling (using GATK 4.1.7 /Haplotype caller). Generated files were then processed with Tapestri Insights v3.1 (Mission Bio) in collaboration with Patnaik Lab as described in detail.¹²⁰

[0065] Third, the transcripomic alterations in the SAC pathway associated with leukemic transformation will be defined. The primary goal of this experiment is to quantify transcriptomic alterations in the SAC components associated with progression of TP53^mut CClIS. 1 x10⁷ cryopreserved BMMC from progressive and non-progressive TP53^mut CUS (n- 15 each at 3 timepoints) will be used. Enriched CD34+/CD38- HSCs (expected 1 -2% of the population) will be isolated as above and total RNA (25-30 ng) will be isolated using standard protocol (Aurum Total RNA Kit, Bio-Rad). Multiplex real time PCR will be performed using a customized version of Cell Cycle Tier2 Panel, PrimePCR™ Assays, Bio-Rad) that includes the known SAC components and analyzed using CFX Maestro Software using the standard configurations. Most differentially expressed genes between the two cohorts will be validated using individual quantitative RT-PCR using different primers.

[0066] Next, the efficacy of TTKi in selectively eliminating TP53^mut clones will be determined. The primary endpoint of this experiment is to quantify the reduction in the percentage of TP53^mut cells following treatment with CFI-402257 or other TTKi as described herein. The secondary outcome is to exclude the induction of aneuploidy in TP53^wt cells via karyotyping. 3x10⁷ cryopreserved BMMC from progressive TP53^mut CCUS cases (n=7) before leukemic transformation will be obtained. Cells will be cultured in the presence of vehicle control or increasing concentration of 10 nM, 30 nM, and 100 nM CFI-402257, or other TTKi, for 72 hours. After washing, the cells will be plated in MethoCult for CFU assay as described and total number of colonies determined at 10 days. Next, up to 100 colonies will be harvested and washed to remove the methylcellulose. Colonies will be sequenced for TP53 and the proportion of colonies with TP53^mut will be determined and compared to vehicle control. Next, TP53^wt colonies will further be evaluated using karyotypic analysis as described.¹²¹

[0067] For the first and second experiments in the Additional Approaches, the standard statistical analysis is not applicable given the high-throughput nature of the technique. The use of case-control and sequential sampling approach allows to track dynamic changes in SNV/CNA, as the subsequent samples will be compared to the baseline sample from the same patient. RT-PCR will be performed in triplicates and mean will be used for subsequent analysis. Fold-change compared to control will be used for reporting and Benjamini- Hochberg False Discovery Rate will be used to correct for multiple testing. Inclusion of 15 patients/group will allow power (1 -p) of 90% with two-sided significance level (a) of 5% to assess for >1.5-fold change compared to controls. CFU assays will be performed in technical triplicate with the average used for analysis. Assuming 30% variance and 50% decrease in TP53^mut colonies using the lowest concertation, inclusion of 7 paired cases before and after treatment will allow detection of difference with 90% power and 2-sided a of 5%.

[0068] A significantly higher proportion of progressive TP53^mut CCUS are expected to harbor CNA of chromosomes 5 and/or 7 prior to leukemic transformation compared to the non-progressive cases. Single-cell sequencing will confirm colocalization of biallelic TP53^mut with chromosome 5 and/or 7 alterations in the HSC and progenitor cells before leukemic transformation. Concurrently performed WGS will confirm TP53^mut VAF and the number of co-mutations to be poor surrogates of the transformation risk. The third set of experiments will demonstrate alterations of the expression of the SAC genes, including the overexpression of TTK, in progressive CCUS that will be validated using the single-gene assay. Finally, in the fourth set of experiments, paired analysis of TP53^mut CCUS samples before and following treatment with CFI-402257, or other TTKi as described herein, will demonstrate dose-dependent decrease in percentage of TP53^mut colonies — but not TP53^wt colonies — compared to vehicle control, suggesting the selective activity of CFI-402257 and other TTKi in TP53^mut CCUS.

[0069] For CNA analyses, the tissue-normal comparator approach is not feasible due to the unavailability of the paired normal (e.g., skin biopsy). The ‘tumor only’ WGS approach is validated and identifies 100% CNA detectable by other cytogenetic methods and is expected to reduce analytical complexity and cost without compromising the conclusions.¹¹¹ For single-cell analyses, the proposed targets of interest are based on the prevalent knowledge of myeloid biology as well as the preliminary observations. However, given the inclusion of diverse primary malignancies and treatments received, it is likely that novel targets of interest may emerge. Thus, the concurrent use of WGS will allow a non-biased approach and inform the choice of genes/CNA to be included in the single-cell sequencing panel. The choice of multiplex RT-PCR over single-cell or bulk RNA sequencing (RNA-seq) was based on the predicted RNA availability of <50 ng (as opposed to >100 ng needed for RNA-seq) from TP53^mut CCUS BMMC. The choice of genes in the customized panel is based on the current knowledge of SAC and other cell cycle components and therefore is subject to bias. Inclusion of up to 96 cell cycle genes, however, will allow for a comprehensive analysis of the cell cycle and SAC. Appropriateness of the approach is further validated given that a vast majority of the differentially expressed genes in TP53^mut cancers belonged to the cell cycle/SAC pathways.^{58 59} Based on the current understanding of the mechanism of TTKi, it is expected that CFI-402257 and other TTKi as described herein will selectively eliminate aneuploid downstream to TP53. Therefore, while conceivable, it is unlikely to induce undesired aneuploidy in TP53^wt cells. The available safety data from murine and early human studies strongly argues against the possibility. CFI-402257 was associated with typical side effects including cytopenia, but no second malignancies have not been reported.^13-15’ ⁵⁶’ ⁶⁰’ ⁹⁷’ ¹²² Nevertheless, induction of aneuploidy in TP53^wt cells is critical and will be carefully excluded as above.

[0070] Example 2: Evaluation of mechanisms of TTKi-mediated anti-leukemic activity in TP53^mut MN.

[0071] Overexpression of TTK, anti-leukemic activity of a highly selective TTKi CFI- 402257, and mechanisms thereof have been evaluated and will be confirmed in a large cohort of newly diagnosed TP53^mut MN. The anti-leukemic activity of additional TTKi as described herein will also be confirmed. A novel model will be used to quantify the impact of TP53 and ploidy status on TTKi-mediated apoptosis. Using a combination of targeted and unbiased methods, how TTKi restores sensitivity to venetoclax in highly venetoclax resistant TP53^mut MN will be characterized.

[0072] Example 2 validates senescent-associated myeloid cytokines, aneuploidy, and SAC dysfunction as the drivers for leukemogenesis and identify strategies to prevent and treat TP53^mut MN.

[0073] TKK is overexpressed in all high-risk MN, including TP53^mut MN, AML with CK, and relapsed/refractory AML. Analysis of the Leukemia MILE93 database showed that TTK is also overexpressed in MDS (n=228) compared to healthy controls (n=989, P<0.001 , Figure 2A), though samples were not further stratified by TP53 status. Analysis of the BEAT AML database⁹⁴ (n=591 , 41 .6% diploid, 35.9% non-CK, 22.7% CK) showed TTK expression to be progressively higher with increasing chromosomal abnormality burden (Figure 2B, C). TTK was overexpressed in TP53^mut (n=27) compared to TP53^wt (n=1 19) AML (P=0.0008, Figure 2D). TTK was overexpressed in relapsed/refractory AML (n=2018) compared to newly diagnosed AML (n=2218, Figure 2E). Finally, screening of MN cell lines showed that TTK is overexpressed in TP53^mut compared to TP53^wt MN (Figure 2F).

[0074] Taken together, the Figure 2 data demonstrates that TTK is overexpressed in all high-risk MN, including TP53^mut MN, AML with CK, and relapsed/refractory AML. Screened myeloid cell lines are representative of TP53^mut MN and will facilitate mechanistic research.

[0075] An orally bioavailable, highly specific, and potent inhibitor of TTK (CFI-402257) has been previously described.^{95 96} Toxicology studies of CFI-402257 in rodents and dogs showed no evidence of overt toxicity.⁹⁶ Importantly, CFI-402257 has shown promising activity against colon, ovarian, hepatocellular carcinoma, and breast cancer and is being studied in various solid malignancies (NCT05251714, NCT03568422, and NCT02792465).¹³’ 18, 56, 60, 97 pi_rst, CH-402257 was assessed to determine activity against TP53^mut AML. CFI- 402257 induced dose-dependent apoptosis and reduced clonogenic potential in bone marrow mononuclear (BMMC) of TP53^mut AML (n=3) but not from BMMC of TP53^wt AML (n=5) or from healthy donors (n=4, Figure 3). Next, it was confirmed that TP53^mut myeloid cell lines represent a valid model for mechanistic experiments by confirming dose-dependent inhibition of cell proliferation and clonogenic potential (representative data for TP53^mut U937 and TP53^wt OCI-AML3 shown, Figure 4A, B). CFI-402257 reduced clonogenic potential of all TP53^mut cell lines tested (representative U937 data, Figure 4C). Available studies suggest diverse — and somewhat contrary — mechanisms of anti-cancer activity of TTK inhibitors.¹³’ ¹⁵’ ^{18 56} TP53^mut myeloid cell lines were treated with CFI-402257 with GI25 (sat 48 hours) of CFI- 402257. Long-term treatment with GI25 dose induced profound aneuploidy and apoptosis in the absence of appreciable induction of polyploidy (representative U937 data, Figure 4D-E). Finally, additional TTK inhibitor MPI-0479605 showed similar anti-leukemic activity, suggesting that the effect seen is secondary to TTK inhibition and not an off-target effect (data not shown). These results suggest overall robust anti-leukemic activity following TTKi.

[0076] Highly venetoclax resistant TP53^mut U937 cells were treated in the absence or presence of GI25 dose (<15% apoptosis as single-agent) of CFI-402257 for 36 hours. Cotreatment led to >10-fold decrease (3665 vs. 348 nM, P<0.001 ) in GI50 for venetoclax, suggesting restoration of sensitivity (Figure 5). Therefore, at a low concentration that does not induce significant apoptosis on its own, TTKi restored sensitivity to venetoclax in highly VEN-r myeloid cells. These results suggest that at low concentrations that do not induce significant apoptosis on its own, TTKi restored sensitivity to venetoclax in highly VEN-r myeloid cells. Collectively, these results highlight SAC dysfunction as a therapeutic vulnerability in TP53^mut AML and suggest that TTKi may demonstrate anti-leukemia activity against TP53^mut MN both as a single-agent and in combination with BCL2-inhibitor venetoclax.

[0077] Additional Approaches

[0078] While the use of mitotic inhibitors in MN has historically been limited, novel agents targeting SAC dysfunction are currently being investigated. Inhibition of polo-like kinase (PLK)-4, a master regulator of centriole biogenesis, is effective in AML.⁵⁵’ ¹²³’ ¹²⁴ In TP53^mut AML, PLK-4 inhibition exerts anti-leukemia effect primarily via senescence, and the role of apoptosis remains to be clarified.¹²⁴’ ¹²⁵ Further, the induction of polyploidy was thought to be critical to PLK4 inhibitor-mediated apoptosis.¹²⁵ In contrast, TTK inhibitor CFI-402257 briskly induced apoptosis in TP53^mut AML that was highly disproportional to polyploidy induction. Finally, whether TTKi restores sensitivity to routinely used AML treatment venetoclax, and mechanism thereof, have not been studied. [0079] First, TTK overexpression will be validated, and the efficacy of CFI-402257 and other TTKi described herein will be investigated in a large cohort of TP53^mut MN. Next, a combination of genetic and pharmacological approaches will be used to confirm that the antileukemic effects seen following treatment with CFI-402257 or other TTKi are indeed due to TTKi and not off-target effects. Third, whether TP53^mut non-CK patients would benefit from TTKi is not known. Therefore, TTKi effects will be studied, stratified by TP53- and ploidy status. Next, TTK exerts proliferative and anti-apoptotic effect via activation of the PI3K-AKT- mTOR pathway.¹⁸ While a majority of AML harbor constitutive activation of PI3K/AKT signaling, targeting the pathway has been challenging due to the paradoxical activation.¹²⁶ Therefore, demonstration of TTKi-mediated inhibition of the PI3K-AKT-mTOR signaling may allow synergetic strategy design. Finally, a proposed mechanism of TTK-mediated cell survival independent of its role in the SAC involves mitochondrial localization — suggesting its interaction with BCL2 pathway proteins.¹²⁷ Whether TTKi leads to mitochondrial outer membrane permeabilization (MOMP) will be assessed, which would restore sensitivity in VEN-r TP53^mut MN.

[0080] First, TKK overexpression and the efficacy of TTKi in TP53^mut MN patient samples will be validated. The primary goals of these experiments are to validate (i) TTK overexpression and (ii) anti-leukemia activity of CFI-402257 and other TTKi in newly diagnosed TP53^mut MDS and AML. The secondary goal is to confirm that anti-leukemia activity exerted by CFI-402257 and other TTKi is secondary to TTKi. Cryopreserved BMMC from newly diagnosed TP53^mut MDS and AML (n=15 each) and age- and sex- matched healthy donors (n=15) will be obtained. TTK mRNA and protein expression will be measured using qRT-PCR and western blot respectively and compared to healthy donor BMMC. Next, BMMC from healthy donors and TP53^mut MN (n=10 each) will be treated with vehicle control or increasing concentrations of CFI-402257 or other TTKi for 36 hours and apoptosis and clonogenic potential quantified as described. The expression of TTK will be correlated to apoptosis and CFU potential. Other selective TTKi will be evaluated [empesertib (Bayer 1161909), ¹²⁸ BAY-1217389,¹²⁸ NMS-715,¹⁶ BOS172722, ¹²⁹ and MPI-0479605^{15 62}] in TP53^mut/nu" cell lines using cell proliferation, apoptosis, and clonogenic potential. Next, to confirm that inhibition of TTK is responsible for the anti-leukemic activity in MN, the above experiments will be repeated following small hairpin (sh)-RNA mediated knockdown of TTK in MOLM-16 cells that are TP53^mutand robustly expresses TTK.⁶³

[0081] Next, the impact of TP53-status and genomic instability on the efficacy of TTKi will be characterized. The primary goal of these experiments is to distinguish TTKi-mediated anti-leukemic activity stratified by TP53 and ploidy status. For this experiment, TP53- isogenic cell lines of MV-4-11 was created using CRISPR-Cas9 mediated knockout (TP53KO) using the lentiviral approach. Briefly, TP53 or no target control (NTC) single guide (sg)-RNAs were designed, ligated to LentiCRISPRv2-mCherry plasmid, and co-transfected into HEK293T cells with the packaging plasmids pMD2.G and psPAX2 (AddGene #12259 and #12260). Lentiviral production and transduction was performed using standard protocol.¹³⁰ Following 5 days of culture, mCherry was assessed using fluorescence microscopy and sorted via fluorescence-activated cell sorting (FACS). Similarly, to create the isogenic TP53 cell line using the knockdown approach, small hairpin (sh)-RNA targeting TP53 (Mission shRNA), and 1 non-target control (NTC) will be cloned into pLKO.1_U6- shRNA: hPGK-Puro-CMV-tGFP (Millipore Sigma) will be used. Isogenic parental and TP53- deficient MV-4-1 1 cells will be used for subsequent experiments. To generate an induced model of polyploidy, near euploid MV-4-11 cells will be treated with 0.75 pM cytochalasin D (ThermoFisher) for 18 hours.¹⁵ Cells will be washed and rested for 24 hours. Induction of polyploidy will be confirmed using Hoechst 33342. All 4 groups will be treated with increasing concentrations of CFI-402257 or other TTKi for 1 -3 days and cell proliferation, apoptosis, and CFU potential assessed as above. To validate that the readouts following CFI-402257 or other TTKi are indeed secondary to TTKi, the experiments will be repeated using TTK^KD/TTK^KO approaches. Finally, the preliminary data suggests a novel mechanism of CFI- 402257- or other TTKi-mediated apoptosis in the absence of polyploidy induction. These findings will be confirmed in the panel of TP53^mut/nu" cell lines (MOLM-16, LI937, and THP-1). Following treatment with 0, 10, or 100 nM CFI-402257 or other TTKi for 72 hours, induction of aneuploidy and polyploidy will be determined using Hoechst staining and the 2N, 4N, >4N cells will be isolated using FACS. Early and late apoptotic cells will be quantified using activated BAX/BAK and cytochrome C release, MOMP, and caspase cascade activation by flow cytometry.

[0082] Third, the mechanism of TTKi-mediated anti-leukemic activity in TP53^mut MN will be determined. The primary goal of this experiment is to demonstrate that TTKi mediates antileukemic activity via the inhibition of PI3K-AKT signaling, resulting in decreased glycolysis. To that end, TP53^mut MN cells will be assessed to determine whether TTKi-mediated antileukemic activity is secondary to the dose-dependent inhibition of PI3K-AKT as measured by decreased phosphorylation and caspase activation will be evaluated.¹⁸ Inhibition of glycolysis will be demonstrated via pre- and post-treatment measurements of the OCR and the ECAR. Hypothesis-driven assessment of the PI3K-AKT pathway will be complemented by an unbiased whole transcriptomics approach. CD34⁺ enriched BMMC of treatment naive TP53^mut MDS and AML (n=7 each) will be obtained and treated with IC50 of CFI-402257 or other TTKi for O, 6, and 12 hours. Total RNA will be isolated and RNA-sequencing experiment and analyses performed and analyzed.⁴⁷’ ¹³¹ Most differentially expressed genes will be validated orthogonally using qRT-PCR and western blot in a larger cohort (n=15). Critical nodes in the most significant pathways enriched in the gene set analysis⁴⁷ will be comprehensively interrogated for activation/inhibition following CFI-402257 or other TTKi treatment and validated using TTK^KD and TTK^KO approaches.

[0083] Next, the mechanism of TTKi-mediated resensitization to venetoclax in TP53^mut MN will be determined. The primary outcome of this experiment is to confirm that TTKi restores sensitivity to venetoclax in VEN-r TP53^mut AML. The secondary outcome is to characterize the mechanism of restoration of venetoclax sensitivity in VEN-r TP53^mut AML. First, TTK mRNA and protein expression will be measured in VEN-r TP53^mut AML and compared to newly diagnosed TP53^mutAML (n=15 each). Next, TP53^mut AML BMMC will be treated with increasing concentrations of venetoclax for 36 hours in the presence or absence of GI25 concentration of CFI-402257 or other TTKi, and assessed for induction of apoptosis and CFU potential. Next, to characterize the mechanisms of CFI-402257- or other TTKi- mediated resensitization of TP53^mut VEN-r cells, U937 cells will be treated with increasing concentration of venetoclax in the absence or presence of GI25 of CFI-402257 or other TTKi for 0, 2, 6, and 12 hours. Upon completion of the experiment, a comprehensive evaluation of mitochondrial pathway proteins, MOMP, BAX and BAK activation (G317-2, Fisher Scientific), and Cytochrome c Release Assay (Abeam, cat# ab6531 1 ) will be performed. Finally, BCL-2 independent effect on the inhibition of glycolysis and oxidative phosphorylation will be studied comprehensively.

[0084] For the first set of experiments in the Additional Approaches, assuming 1 .5-fold higher expression of TTK in TP53^mut MDS and AML compared to matched controls, the inclusion of 15 cases/subgroup will allow power (1 -p) of 90% with two-sided significance level (a) of 5%. Apoptosis and CFU assays will be performed in technical triplicate with the average used for analysis. Based on preliminary observations, addition of CFI-402257 or other TTKi will result in at least 25% higher apoptosis and reduced clonogenic potential in TP53^mut MN compared to matched controls. Therefore, inclusion of 10 cases/group will allow power (1 -p) of 90% with two-sided significance level (a) of 5%. Cell line experiments will be performed in triplicates and each experiment will be performed at least three times and the representative set will be used for subsequent studies, inference, and publication.

[0085] Collectively, these experiments will establish efficacy of CFI-402257 or other TTKi and the mechanism thereof. Selective apoptosis and reduced clonogenic potential in TP53^mut MDS and AML samples compared to healthy donors will demonstrate therapeutic window and inform subsequent dosing strategy. Understanding the mechanisms of activity as single agent as well as in combination with venetoclax will help devise synergetic strategies and identify patients at risk for developing resistance to TTKi. TP53^mut MN are considered a single entity⁵ and share biological characteristics regardless of the bone marrow blast percentage.³ Therefore, differences in TTK expression and/or sensitivity to TTKi based on BM blast are not expected. In the event of differential expression and/or sensitivity, subsequent analyses will be performed stratified according to morphological subcategories.⁵ Given the high specificity of CFI-402257, TTK^KO and TTK^KD are expected to confirm the antileukemic activity seen with the panel of TTKi. Discrepancy in anti-leukemic activity of CFI- 402257 or other TTKi and TTK knockdown will suggest an off-target effect of CFI-402257 or other TTKi that will be interrogated using broad profiling of kinases with a particular emphasis on cell cycle regulators.⁵⁶ Third, TTKi is expected to act downstream to TP53 and aneuploidy and not the TP53-status will be determinant of its efficacy, suggesting its use in TP53^mut CK and a minority of TP53^wt MN with CK.³’ ⁵¹ • ¹⁰⁹ In MN, VEN-r develops via a wide array of mechanisms with upregulation of anti-apoptotic BCL2 family member proteins and metabolic reprogramming appear to the best common mechanisms.⁶⁴’ ¹³²’ ¹³³ In the event that neither of these mechanisms are involved in the restoration of venetoclax sensitivity, RNA- sequencing approach will be performed. Finally, while the preliminary evidence strongly suggests apoptosis as the most likely mechanism of anti-leukemic activity. In the event that apoptosis is not the dominant mechanism of cell death, non-apoptotic mechanisms including GAS-STING activation,¹³ senescence induction,¹²⁴ necroptosis, and autophagy will be explored.

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Claims

What is claimed is:

1 . A method of treating a subject diagnosed with TP53^mut myeloid neoplasm (TP53^mut MN), said method comprising administering a threonine tyrosine kinase inhibitor (TTKi) to the subject.

2. A method of treating TP53^mut MN in a subject, said method comprising the steps of:

(a) diagnosing the subject with TP53^mut MN; and

(b) administering a threonine tyrosine kinase inhibitor (TTKi) to the subject.

3. A method of increasing genomic stability in a subject, said method comprising the steps of:

(a) diagnosing the subject with TP53^mut MN; and

(b) administering a threonine tyrosine kinase inhibitor (TTKi) to the subject.

4. A method of increasing dysfunction of the spindle assembly checkpoint (SAC) in a subject, said method comprising the steps of:

(a) diagnosing the subject with TP53^mut MN; and

(b) administering a threonine tyrosine kinase inhibitor (TTKi) to the subject.

5. A method of decreasing survival of aneuploidy cells in a subject, said method comprising the steps of:

(a) diagnosing the subject with TP53^mut MN; and

(b) administering a threonine tyrosine kinase inhibitor (TTKi) to the subject.

6. A method of preventing progression of TP53^mut CCUS (Clonal Cytopenia of Undetermined Significance) to TP53^mut MN in a subject, said method comprising administering a TTKi to the subject.

7. A method of restoring venetoclax sensitivity in venetoclax-resistant cells in a subject, said method comprising administering a TTKi to the subject.

8. The method according to any one of the preceding claims, wherein the subject has a complex karyotype.

9. The method according to any one of the preceding claims, wherein the subject has 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomal abnormalities.

10. The method according to any one of claims 8 or 9, wherein the chromosomal abnormality is selected from the group consisting of a chromosomal deletion, duplication, inversion, and translocation.

1 1 . The method according to any one of claims 8-10, wherein the chromosomal abnormalities comprise chromosome 5 abnormalities, chromosome 7 abnormalities, or abnormalities in both chromosome 5 and 7.

12. The method of claim 1 1 , wherein the abnormalities are selected from deletions of chromosomes 5q or 7q.

13. The method of any one of claims 8-10, wherein the TP53^mut phenotype comprises one or more of T125_splice, H179, Y220, R175, R213, G245, R248, R273, and R282 mutations in the TP53 gene.

14. The method according to any one of the preceding claims, wherein the TP53^mut MN is selected from the group consisting of relapsed/refractory MN, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

15. The method according to any one of the preceding claims, wherein the TTKi is selected from the group consisting of CFI-402257, S-81694, BAY-1217389, BOS-172722, CC-671 , OSU-13se, VRN-081569, Empesertib, BAL-0891 , CCT-251455, CFI-401870, MPI-0479605, and NTRC-0066-0

16. The method of claim 15 wherein the TTKi is CFI-402257.

17. The method according to any one of the preceding claims, further comprising administering at least one additional therapeutic agent in combination with the TTKi.

18. The method of claim 17 wherein the at least one additional therapeutic agent is selected from the group consisting of a BCL-2 inhibitor, intensive chemotherapy (doxorubicin, daunorubicin, idarubicin, cytarabine, azacitidine, decitabine), and a stem cell transplant.

19. The method of claim 18, wherein the additional therapeutic agent is a BCL-2 inhibitor selected from the group consisting of venetoclax, navitoclax, obatoclax, and oblimersen sodium.

20. The method of claim 19 wherein the BCL-2 inhibitor is venetoclax.