WO2019229001A1

WO2019229001A1 - Compounds for the treatment of jak2-v617f-associated cmn

Info

Publication number: WO2019229001A1
Application number: PCT/EP2019/063683
Authority: WO
Inventors: Thomas Fischer; Bärbel EDELMANN-STEPHAN
Original assignee: Otto-Von-Guericke-Universität Magdeburg
Priority date: 2018-05-30
Filing date: 2019-05-27
Publication date: 2019-12-05

Abstract

The present disclosure provides novel compounds for use in therapy of JAK2-V617F-associated chronic myeloproliferative neoplasia (CMN) and for use in therapy or prophylaxis of disorders or syndromes associated therewith, in particular splenomegaly and increased risk of thrombosis.

Description

Compounds for the treatment of JAK2-V617F-associated CMN

DESCRIPTION The present invention provides novel compounds for use in therapy of JAK2-V617F- associated chronic myeloproliferative neoplasia (CMN) and for use in therapy or prophylaxis of disorders or syndromes associated therewith, in particular splenomegaly and increased risk of thrombosis.

Chronic myeloproliferative neoplasia (CMN) is a malignant hematopoietic disease characterized by excessive proliferation of one or more myeloid cell lineages as erythrocytes, platelets or leukocytes. CMN comprises several sub-entities including polycythemia vera (PV), essential thrombocytosis (ET), primary myelofibrosis (PMF) and others. Genomic analysis of CMN revealed an activating somatic point mutation of the JAK2 (Janus kinase 2) gene (JAK2-V617F) in the majority (95%) of PV patients and in 50% of ET and PMF patients, respectively. JAK2-V617F mutated CMN (PV, ET and PMF) is driven by clonal proliferation of myeloid cells. Patients may develop an inflammatory syndrome consisting of fever, night sweats and elevated serum proinflammatory cytokine levels. Venous and arterial thrombosis is the major cause of morbidity and mortality in PV and ET patients. Strikingly, the thrombotic risk in these patients is far in excess of that seen in patients who have secondary crythrocytosis or thrombocytosis. This indicates that disease-intrinsic factors play a major role. Indeed, it has been reported that JAK2-V617F leads to abnormal function of erythrocytes and platelets.

Furthermore, the occurrence of extramedullary hematopoiesis in the spleen of JAK2- V617F positive CMN is associated with abnormal trafficking of hematopoietic cells including clonal hematopoietic stem and progenitor cells. Abnormal interaction of JAK2-V617F activated granulocytes and monocytes with splenic cells may contribute to an inflamed microenvironment, which may stimulate extramedullary hematopoiesis and thus splenomegaly. Although the exact molecular process is not entirely clear, it has already been proposed that potential mechanisms likely include differential response to the unique splenic microenvironment of JAK2-V6l7F-positive disease. Along this line, a striking change in relative proportion of immune cells as granulocytes, monocytes, B- cells and T-cells in the spleen of murine JAK2-V6l7F-positive disease has been described.

Clinical studies using JAK kinase inhibitors have shown improvement in symptoms splenomegaly, systemic inflammatory symptoms and thus overall survival. However, in many patients the success of such JAK kinase inhibitors in clinical therapy is not durable. A considerable proportion of patients show progressive disease within 2-3 years. In addition, clinical responds are incomplete in many patients. Moreover, some of the JAK kinase inhibitors are rather unspecific and bear the risk of negative side effects in the patient. Thrombotic events are the major cause for morbidity and mortality in JAK2-V617F positive PV and ET. Neutrophils, monocytes, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. Neutrophils are recruited during the early phase of venous thrombosis to the intact endothelial surface, constitute the major leukocyte subset and play a predominant role. The process of neutrophil attachment to the endothelium and platelet-neutrophil interaction is predominantly mediated by selectin and integrin activation. Despite recent advances in understanding the pathophysiology of JAK2-V617F induced CMN, the molecular mechanism that triggers the marked pro-thrombotic state, the primary cause of morbidity and mortality in patients with PV and ET, remains elusive. Integrins are heterodimeric proteins made up of alpha and beta subunits. At least 18 alpha and eight beta subunits have been described in mammals. The beta-subunits each associate with an alpha-subunit to form integrin complexes (heterodimers). Integrin family members are membrane receptors involved in cell adhesion and recognition in a variety of processes including embryo genesis, hemostasis, tissue repair, immune response and metastatic diffusion of tumor cells. Integrins may link the actin cytoskeleton with the extracellular matrix and they transmit signals bidirectionally between the extracellular matrix and cytoplasmic domains. Endothelial cells express the betal- and beta2-integrin subunit ligands ICAM1 and VCAM1 which are particularly abundant under inflamed conditions. Betal- and beta2-integrins (ITGB1 and ITGB2), also known as CD29 and CD 18, respectively, are cell surface receptors that in humans is encoded by the ITGB1 and ITGB2 gene, respectively. CD29 associated together with the alpha-4 subunit (ITGA4) forming VLA-4 while CD 18 when associated with the alphaL subunit (IT GAL) is called LFA-l.

Interestingly, tissue-specific expression of JAK2-V617F in megakaryocytes, and thus in platelets, failed to show significant differences in occlusion time between WT and Pf4- Cre/FFl mice when using FeCl3 occlusion assays of the carotid artery. Moreover, membrane expression of integrin beta3, alpha lib, betal, and alpha2 was unchanged in resting platelets and no significant differences were observed in the activity of the platelets as tested by P-selectin expression and activated alphallbbeta3 levels. These results point to a minor role of platelets in JAK2-V617F induced pathologic thrombus formation. The reticulocyte/erythrocyte compartment of JAK2+/+ and JAK2+/VF mice express very low levels of CD29 (VLA-4) and of CD 18 (LFA-l) which are barely detectable and apparently unchanged in JAK2+/VF mice. Thus, this finding argues against a major role of betal- and beta2-integrins expressed on erythrocytes when inhibiting VLA-4/beta2 in a JAK2-V6l7F-induced thrombosis model.

The technical problem underlying the present invention is the provision of novel and improved methods and means for the safe and treatment of JAK2-V6l7F-associated chronic myeloproliferative neoplasia (CMN) and treatment or prophylaxis of disorders or syndromes associated with JAK2-V6l7F-associated chronic myeloproliferative neoplasia (CMN) in an animal or human individual.

The technical problem is fully solved by the provision of a pharmaceutical composition which comprises: an inhibitor of integrin betal-subunit (IGTB1) and/oran inhibitor of integrin beta2-subunit (IGTB2) or inhibitors for the corresponding alpha-subunit of the integrin receptor or substantially consist thereof. In a 1 st aspect, there is provided a pharmaceutical composition for use in therapy of JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) and of disorders or syndromes associated with JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) in an animal or human individual, the pharmaceutical composition comprising an inhibitor of betal-integrin and/or an inhibitor of beta2-integrin.

In a 2nd aspect, there is provided the composition of said lst aspect, comprising an inhibitor of betal -subunit of betal-integrin and/or an inhibitor of beta2-subunit of beta2-integrin.

In a 3rd aspect, there is provided the composition of aspect 1 or aspect 2, comprising an inhibitor of alpha4-subunit of betal-integrin and/or an inhibitor of beta2-subunit of beta2-integrin.

In a 4th aspect, there is provided the composition of any one of the preceding aspects 1 to 3 wherein the betal -integrin-inhibitor is a first compound that specifically binds to the betal -subunit or a compound that specifically binds to the alpha4-subunit and wherein the beta2 -integrin-inhibitor is a second compound that specifically binds to the beta2-subunit.

In a 5th aspect, there is provided the composition of any one of the preceding aspects 1 to 4, wherein the integrin-inhibitor is selected from monoclonal antibodies specifically binding to the betal- or beta2-subunit of said betal- or beta2-integrin or to the corresponding alpha-subunit of said betal - or beta2-integrin.

In a 6th aspect, there is provided the composition of any one of the preceding aspects 1 to 5, wherein the integrin-inhibitor is selected from ligands and antagonists to the betal - or beta2-subunit of said betal- or beta2 -integral or to the corresponding alpha-subunit of said betal - or beta2 -integral In a 7th aspect, there is provided the composition of any one of the preceding aspects 1 to 6, further comprising an inhibitor of Rapl-GTPase. In an 8th aspect, there is provided the composition of any one of the preceding aspects 1 to 7, wherein the JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) is essential thrombocythcmia (ET), polycythemia vera (PV) or primary myelofibrosis (PMF). In a 9th aspect, there is provided the composition of any one of the preceding aspects 1 to 8, wherein the JAK2 V6l7F-associated disorder or syndrome is JAK2 V617F- associated splenomegaly, JAK2 V6l7F-associated increased risk of thrombosis and JAK2 V6l7F-associated inflammatory response syndrome.

In a lOth aspect, there is provided an in vitro method for inhibiting adhesion of granulocytes, the method comprising the step of: contacting granulocytes with the composition of any one of aspects 1 to 6.

In an 1 lth aspect, there is provided an in vitro method for treating isolated tissue or cells having symptoms of JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN), the method comprising the step of: contacting the isolated tissue or cells with the composition of any one of aspects 1 to 6.

In a l2th aspect, there is provided the method of aspect 11, wherein the isolated cells are selected from JAK-V6l7F-cells.

In a 13th aspect, there is provided the method of aspect 11 or aspect 12, wherein the isolated cells are leukocytes, selected from monocytes and granulocytes. More particular, according to the invention there is provided a pharmaceutical composition for use in therapy of JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) and of disorders or syndromes associated with JAK2 V617F- associated chronic myeloproliferative neoplasia (CMN) in an animal or human individual, the pharmaceutical composition comprising: (1) an inhibitor of betal- integrin, and (2) an inhibitor of beta2-integrin, in particular in a pharmaceutically acceptable carrier. The pharmaceutical composition of the invention is used in medicine. The pharmaceutical composition of the invention is thus used in a method for the treatment of JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) and treatment or prophylaxis of disorders or syndromes associated with JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) in an animal or human individual. The method comprises the step of administering the pharmaceutical composition of the invention in a dose effective to suppress or alleviate the disorders or syndromes associated with JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) to an animal or human individual in need thereof. The JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) syndromes are preferably selected from: essential thrombocythcmia (ET), polycythemia vera (PV) or primary myelofibrosis (PMF).

The JAK2 V6l7F-associated disorder or syndrome is preferably selected from: JAK2 V6l7F-associated splenomegaly, JAK2 V6l7F-associated increased risk of thrombosis, JAK2 V6l7F-associated venous and arterial thrombotic events and JAK2 V617F- associated inflammatory response syndrome.

The invention is primarily based on the surprising findings that JAK2-V617F activates intracellular signaling of integrals comprising betal- and beta2-integrin subunits in leukocytes. A blockade of both, betal- and beta2 integral activity or blockade of both, an alpha4 subunit in a betal/alpha4-integrin and beta-2 integrin activity suppresses pathologic thrombus formation in vivo. It has been hypothesized that in JAK2-V617F induced diseases an increased ICAM1 and VCAM1 expression on endothelial cells in combination with abnormal betal- and beta2-activity causes abnormal adhesion of granulocytes to endothelial cells which contributes to pathologic thrombus formation. Furthermore, is has been found that the occurrence of extramedullary hematopoiesis in the spleen of JAK2-V617F positive CMN is associated with abnormal trafficking of hematopoietic cells including clonal hematopoietic stem and progenitor cells and that increased beta-l and beta2-integrin activity in JAK2-V617F positive CMN drives abnormal trafficking and homing of granulocytes and monocytes to the spleen. Blockade of betal- and of beta2 integral activity has been shown to suppress such pathologic splenomegaly.

In the context of the present invention, the phrase“blocking or inhibition of betal - integrin” refers to the blocking or inhibition of an integrin (heterodimer) comprising a betal -subunit. This integrin may be blocked or inhibited by blocking or inhibition of its betal -subunit, but may also be blocked or inhibited by blocking or inhibition of its corresponding alpha-subunit of the integrin dimer. The alpha-subunit of the integrin heterodimer comprising the betal -subunit is in particular an alpha4-subunit. Likewise, the phrase“blocking or inhibition of beta2-integrin” refers to the blocking or inhibition of an integrin (heterodimer) comprising a beta2-subunit. This integrin may be blocked or inhibited by blocking or inhibition of its beta2-subunit, but may also be blocked or inhibited by blocking or inhibition of its corresponding alpha-subunit of said integrin dimer. The alpha-subunit of the integrin heterodimer comprising the beta2-subunit is in particular an alpha-L- subunit or alpha-X-subunit. In one embodiment the composition is comprising an inhibitor of betal -subunit of betal -integrin In another or an additional embodiment the composition is comprising an inhibitor of beta2-subunit of beta2 -integrin. In another or an additional embodiment the composition is comprising an inhibitor of alpha4-subunit of betal -integrin. In another or an additional embodiment the composition is comprising an inhibitor of beta2-subunit of beta2 -integrin. In another or an additional embodiment the composition is comprising an inhibitor of alpha-L-subunit of beta2 -integrin. In another or an additional embodiment the composition is comprising an inhibitor of alpha-X-subunit of beta2- integrin.

In preferred embodiments, the betal- and/or beta2- and/or alpha4-subunit integrin- inhibitor is one or more compound that specifically binds to the betal- and/or beta2- integrin subunit and/or the corresponding alpha-integrin subunit and/or specifically blocks the binding site of the betal- and/or beta2- and/or the corresponding alpha- integrin subunit to inhibit integrin receptor activation. In specific variants thereof the betal -integrin-inhibitor is a first compound that specifically binds to the betal -subunit or a compound that specifically binds to the alpha4-subunit and the beta2-integrin- inhibitor is a second compound that specifically binds to the beta2-subunit.

In preferred embodiments, the composition of the invention comprises at least two distinct compounds, one specific to betal- or its corresponding alpha-subunit at its integrin heterodimer, the other specific to beta2-subunit or its corresponding alpha- subunit at its integrin heterodimer. In a preferred embodiment thereof, the betal -subunit integrin-inhibitor is a first compound that specifically binds to the betal -subunit, the beta2-subunit integrin-inhibitor is a second compound that specifically binds to the beta2-subunit, and the alpha4-subunit integrin inhibitor is an alternative first compound that specifically binds to the alpha4-subunit of the betal -containining integrin In preferred embodiments, the integrin-inhibitor is selected from monoclonal antibodies specifically binding to the betal- or beta2-subunit of said betal- or beta2 -integrin or to the corresponding alpha-subunit of said betal - or beta2 -integrin.

In preferred alternative or additional embodiments, the integrin-inhibitor is selected from ligands and antagonists to the betal- or beta2-subunit of said betal- or beta2- integrin or to the corresponding alpha- subunit of said betal - or beta2-integrin.

More particular, the inhibitor of betal -integrin is preferably selected from: blocking compounds specifically binding to CD29, in particular anti-human CD29 blocking antibody, for example, clone P5D2 #MABl959 from Merck/Millipore, and from VLAbl blocking antibody, in particular anti-human VLAbl blocking antibody, for example, clone 6A267 from (Holzel Diagnostica) or CD29 blocking peptide, for example, # LS-E29653 from LifeSpan BioSciences.

The inhibitor of beta2-integrin is preferably selected from: blocking compounds specifically binding to CD 18, in particular anti-human CD 18 blocking antibody, for example, clone TS 1/18 #302116 from Biolegend, or CD18 blocking peptide, for example, #MBS8237822 from MyBioSource.

The inhibitor of alpha4-subunit of betal -integrin is preferably selected from: blocking compounds specifically binding to CD49d, in particular anti-human CD49d blocking antibody, for example, clone PS/2 from (Holzel Diagnostica) or Natalizumab (Tysabri®) or alpha4 (CD49d) blocking peptide, for example, # MBS 153211 from MyBioSource.

It has been further found that inhibition of small GTPase Rapl (Ras-proximate-l) suppressed abnormal splenic homing JAK2-V6l7F-positive granulocytes and monocytes in vivo without having an apparent effect on cell survival. This finding is in line with the finding that Rapl activation and membrane re-localization is essential for betal-integrin activation. The results suggest that inhibition of betal- or beta2-integrin activity together with inhibition of GTPase Rapl prevents or reduces or suppresses abnormal trafficking and homing of granulocytes and monocytes to the spleen and thus is effective in treatment or prophylaxis of disorders or syndromes associated with JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) in an animal or human individual, in particular splenomegaly.

In preferred embodiments, the composition of the invention thus further comprises an inhibitor of Rapl -GTPase. The inhibitor of Rapl -GTPase is preferably selected from: Famesylthiosalicyl acid (Cayman Chemicals, Sigma Aldrich or Santa Cruz Biotech), Famesylthiosalicyl amide (Cayman Chemicals), GGTI-2147 (Merck/Millipore).

In a further aspect, the present invention also provides a novel method for inhibiting adhesion of granulocytes in vitro, the method comprising the step of: contacting granulocytes with the composition of the invention as described herein.

In another aspect, the present invention also provides a novel method for treating isolated tissue or cells having symptoms of JAK2-V6l7F-associated chronic myeloproliferative neoplasia (CMN), the method comprising the step of: contacting the isolated tissue or cells with the method of the invention as described herein. The isolated cells are preferably selected from JAK2-V6l7F-cells. The isolated cells are in particular leukocytes, preferably selected from monocytes and granulocytes.

The invention is further described and illustrated by the following examples and figures: Figure 1 shows expression levels of betal and beta2 integrins. (A) betal integral (CD29) expression and (B) beta2 integral (CD 18) expression in the myeloid (granulocytes and monocytes) and the lymphoid (T- and B-cells) compartment of 10- 12-week-old JAK2+/+ (n = 10) and JAK2+/VF (n = 11) mice. Data are shown as mean ± SEM. ns, not significant, **P < 0.01, ***P < 0.001 (unpaired, two-tailed Student's t test).

Figure 2 shows that betal and beta2 integrins are activated on murine granulocytes. (A, B) Static adhesion of murine granulocytes isolated from 10-12-week-old JAK2+/+ (n = 5) and JAK2+/VF (n = 5) mice on Fc-free VCAM1 (A) and ICAM1 (B). (C) Soluble VCAM1 (sVCAMl) binding assay of JAK2+/+ (n = 7) and JAK2+/VF (n = 7) murine granulocytes. (D) Soluble ICAM1 (sICAMl) binding assay of JAK2+/+ (n = 7) and JAK2+/VF (n = 8) murine granulocytes. (E) 9EG7 binding of JAK2+/+ (n = 4) and JAK2+/VF (n = 4) murine granulocytes. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, (unpaired, two-tailed Student's t test). Figure 3 shows that inhibition of Rap 1 is blocking adhesion of murine granulocytes. A) Rapl is activated in JAK2+/VF murine granulocytes. Western-blot derived from pooled granulocytes of two 10-12-week-old JAK2+/+ and two JAK2+/VF mice, respectively. (B) Static adhesion of murine granulocytes isolated from 10-12-week-old JAK2+/VF (n = 5) mice on VCAM1 preincubated with 10 mM GGTI-2147 for 30 min. Data shown as mean ± SEM. ***P < 0.01, (unpaired, two-tailed Student's t test).

Figure 4 shows that betal and beta2 integrins play a major role in thrombosis formation. (A) HUVECs were stimulated with different concentration (1; 5 and 10 ng/ml) of TNFD for 24 h. Flow cytometric analysis was performed for CD54 (ICAM1) and CD 106 (VCAM1) expression (n = 6). (B) Partial ligation of inferior vena cava in 10-12- week-old JAK2+/+ (n = 7) and JAK2+/VF (n = 6) mice results in increased thrombus size and dry weight in JAK2-V617F positive mice. Representative images of isolated thrombi (right panel, scale bar 1 mm). (C) JAK2+/VF mice were treated with VLA4/beta2 integrin blocking antibodies (n = 5) or isotype control (n = 4) before partial ligation of inferior vena cava. Representative images of isolated thrombi (right panel, scale bar 1 mm). Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001 (two-way ANOVA with Bonferroni post-test and unpaired, two-tailed Student's t test).

Figure 5 shows that homing of granulocyte and monocytes can be blocked by beta2 integrin or Rapl blockade. (A) Whole bone marrow (WBM) cells of l0-l2-week-old JAK2+/VF or JAK2+/+ mice (carrying the congenic marker CD45.2) were injected into CD45.1 recipient mice. 16 h after injection spleens of recipient mice were evaluated. Data represents number of detected CD45.2-positive granulocytes and monocytes per 106 cells in the spleen (n = l4/group) and are shown as mean ± SEM. *P < 0.05 (unpaired, two-tailed Student's t test). (B) Effect of beta2 integrin blocking antibody treatment (50 pg/m 1 ; 30 min, n = 9) compared to iso type treated cells (n = 10) on the number of detected CD45.2-positive granulocytes and monocytes, respectively per 106 spleen cells 16 h after tail vein injection. (C) Effect of GGTI-2147 treatment (10 gmol/1; 30 min) on the number of detected CD45.2-positive granulocytes and monocytes, respectively per 106 spleen cells 16 h after tail vein injection (-GGTI-2147, n = 10 and +GGTI-2147, n = 9); (0) indicates DMSO control (left panel). Data shown as mean ±

SEM. **P < 0.05, ***P < 0.01 (non-parametric Mann- Whitney U-test).

EXAMPLES

1. Expression levels of betal and beta2 integrins in the hematopoietic system of

JAK2+/VF mice To investigate the effects of physiological JAK2-V617F expression on betal and beta2 integrin abundance, a JAK2-V617F knock-in mouse CMN model was used (36). Floxed heterozygous JAK2-V617F (JAK2+/floxP-VF-floxP) mice were crossed with transgenic VavCre mice (JAK2+/VF) resulting in JAK2-V617F protein expression in hematopoiesis. The resulting phenotype is characterized by an elevated hematocrit and WBC count, splenomegaly and prominent splenic extramedullary hematopoiesis and resemble human polycythemia vera (PV). Expression levels of the two most prominent leukocyte integrins betal and beta2 were analyzed in the myeloid and lymphoid compartment of the bone marrow of JAK2+/+ (Cre+) and JAK2+/VF (Cre+) mice betal (CD29) integrin expression was unchanged in granulocytes of JAK2+/VF mice compared to JAK2+/+ (Figure 1A). Interestingly, in monocytes, T-cells and B-cells expression of betal integrins was upregulated featuring a most prominent effect in monocytes (Figure 1A) and indicating a differential regulatory mechanism of betal integrin expression in these cell types. In parallel, the expression levels of beta2 integrins (CD 18) were found to be increased in granulocytes, monocytes and T-cells but not in B-cells (Figure 1B).

2 betal and beta2 integrin activity is enhanced in primary murine JAK2+/VF granulocytes and is dependent on Rapl-GTPase activity

Using primary granulocytes isolated from the bone marrow of JAK2+/+ or JAK2+/VF mice, static adhesion assays on VCAM1 or ICAM1 (ligand for beta2 integrins) were performed. Increased adhesion of JAK2+/VF granulocytes was detected on VCAM1 as well as on ICAM1 (Figure 2A, B). Soluble ligand binding assays for VCAM1 (sVCAMl) and ICAM1 (sICAMl) revealed increased binding to JAK2+/VF granulocytes as compared to the JAK2+/+ cells (Figure 2C, D). To analyze the conformation status of betal integrins on granulocytes in JAK2+/VF and JAK2+/+ mice, 9EG7 antibody binding was tested (Figure 2E). We found that JAK2+/VF granulocytes showed increased binding of 9EG7 antibody indicating that betal integrins have shifted from the bent, low affinity status to an open, high-affinity conformation.

3. Rapl is constitutive activated in JAK2+/VF granulocytes Having demonstrated that betal and beta2 integrin activity is increased in JAK2+/VF granulocytes, we evaluated inside-out signaling of integrins. Analysis of JAK2+/+ and JAK2+/VF granulocytes demonstrated an increased activation of Rapl by JAK2-V617F mutation in mice (Figure 3A). Incubation with the geranylgeranyltransferase inhibitor GGTI-2147, which blocks posttranslational modifications of Rapl required for activation and translocation to the plasma membrane (Schmid MC, Franco I, Kang SW, Hirsch E, Quilliam FA, Vamer JA. PI3 -kinase gamma promotes Rap la-mediated activation of myeloid cell integrin alpha4betal, leading to tumor inflammation and growth. PloS one. 20l3;8(4):e60226. doi: 10.1371 /journal. pone.0060226), revealed a strong decrease in static adhesion on VCAM1 (Figure 3B). This suggests that posttranslational processing of Rapl is required for JAK2-V617F induced integrin activation and adhesion.

4 Blockade of betal and beta2 integrin activity suppresses pathologic thrombus formation in a thrombosis model using JAK2+/VF mice Blood flow restriction triggers the rapid attachment and accumulation of neutrophils and monocytes to the endothelium which is crucial for development of thrombosis. The process of neutrophil attachment to the endothelium and platelet-neutrophil interaction is predominantly mediated by selectin and integrin activation. Endothelial cells express the betal and beta2 integrin ligands ICAM1 and VCAM1 which are particularly abundant under inflamed conditions. To investigate whether inflammatory cytokines, which are commonly elevated in JAK2-V617F induced disease may modulate ICAM1 and VCAM1 expression, human umbilical vein endothelial cells (HUVECs) were tested in response to TNF alpha and IL6 stimulation using flow cytometry. Strong up- regulation of both, ICAM1 and VCAM1, was detected in TNFalpha stimulated cells (Figure 4A), whereas IF6 appears to selectively induce ICAM1 expression (data not shown). Of note, expression of E- and P-selectins was not affected by TNFalpha and IF6 (data not shown). Thus, we hypothesized that in JAK2-V617F induced disease increased ICAM1 and VCAM1 expression on endothelial cells in combination with abnormal betal and beta2 activity may precipitate abnormal adhesion of granulocytes to endothelial cells contributing to pathologic thrombus formation. To study the role of betal and beta2 integrins in this process, we employed thrombosis model of the inferior vena cava (IVC). Thrombosis was induced in JAK2+/+ and JAK2+/VF mice by partial ligation of the inferior vena cava and 4 h thereafter thrombus size and dry weight of the thrombi were analyzed (Figure 4B). A significant increase in thrombus size and dry weight was observed in JAK2+/VF mice as compared to the JAK2+/+ mice confirming previous reports on accelerated thrombosis in JAK2-V617F mice (Famrani F, et al. Hemostatic disorders in a JAK2V6l7F-driven mouse model of myeloproliferative neoplasm. Blood. 2014; 124(7): 1136-1145. doiTO.l l82/blood-20l3-l0-530832). Next, we determined the relative contribution of betal and beta2 integrin activity to JAK2- V617F induced thrombus formation. JAK2+/VF mice were intravenously injected either with a combination of blocking antibodies against VLA-4 and beta2 integrins or with the corresponding isotype controls (Figure 4C). JAK2+/VF mice treated with VLA- 4/beta2 integrin blocking antibodies showed dramatically reduced thrombus dry weight and size as compared to the isotype control injected JAK2+/VF mice. Thus, upon VLA- 4/beta2 integrin blockade, JAK2+/VF mice displayed similar thrombus weight and size as observed in JAK2+/+ mice without VLA-4/beta2 integrin blockade. Together, these results uncover a crucial role of betal/beta2 integrin over-activation in pathologic thrombus formation in JAK2-V617F driven disease.

5. Activated betal and beta2 integrins of JAK2-V617F positive granulocytes stimulate abnormal trafficking to the spleen in vivo

In JAK2-V617F positive disease from mice and humans, abnormal accumulation of mature myeloid cells characterized by expression of Gr-l and CDl lb and extramedullary granulopoiesis within the splenic cords has been observed (Bumm TGP, et al. Characterization of murine JAK2V6l7F-positive myeloproliferative disease. Cancer research. 2006;66(23):l 1156-11165. doi:l0.l l58/0008-5472.CAN-06-22l0; Prakash S, Hoffman R, Barouk S, Wang YL, Knowles DM, Orazi A. Splenic extramedullary hematopoietic proliferation in Philadelphia chromosome-negative myeloproliferative neoplasms: Heterogeneous morphology and cytological composition. Modem pathology: an official journal of the United States and Canadian Academy of Pathology, Inc. 2012;25(6):815-827. doi:l0.l038/modpathol.20l2.33). Because we could demonstrate disturbed expression of VC AM 1 and ICAM1 and altered distribution of granulocytes, we hypothesized that trafficking of myeloid cells into the spleen might be affected. To investigate which cell types preferentially home to the spleen, whole bone marrow cells obtained from JAK2+/+ or JAK2+/VF mice, respectively (carrying the congenic marker CD45.2) were injected into CD45.1 recipients and after 16 h spleens were analyzed for the CD45.2 positive cell population. Strikingly, a higher proportion of granulocytes and monocytes from JAK2+/VF mice homed to the spleen as compared to cells derived from JAK2+/+ littermate controls (Figure 5A). Integrins LFA-l and VLA-4 together with chemokine receptor signaling mediate homing of leukocytes to the spleen (Lo CG, Lu TT, Cyster JG. Integrin-dependence of Lymphocyte Entry into the Splenic White Pulp. The Journal of experimental medicine. 2003;l97(3):353-36l. doi:l0.l084/jem.2002l569). Therefore, bone marrow cells of JAK2+/VF mice were preincubated with a beta2 integrin blocking antibody or the corresponding isotype control before injection into CD45.1 recipients. This analysis revealed a reduced number of granulocytes and monocytes in spleen (Figure 5B). To investigate whether the increased homing ability correlates with constitutive activation of Rap- 1, we preincubated the bone marrow cells with the Rap-l inhibitor GGTI-4721 before injection into recipient mice. Inhibition of Rapl suppressed abnormal splenic homing of injected JAK2-V6l7F-positive granulocytes and monocytes in vivo (Figure 5C). This finding is in line with studies highlighting that Rapl activation and membrane re-localization is essential for betal integrin activation (Medeiros RB, et al. Protein kinase Dl and the beta 1 integrin cytoplasmic domain control beta 1 integrin function via regulation of Rapl activation. Immunity. 2005;23(2):2l3-226. doi:l0.l0l6/j.immuni.2005.07.006). Together the results suggest that increased betal and beta2 integrin activity drives abnormal trafficking and homing of granulocytes and monocytes to the spleen.

6. Methods

6.1 Mouse models

JAK2+/VF mice (Mullally A, et al. Physiological Jak2V6l7F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells. Cancer cell. 20l0;l7(6):584-596. doi:l0.l0l6/j.ccr.20l0.05.0l5). Floxed heterozygous JAK2+/floxP-VF-floxP animals were crossed with Vav-l-Cre transgenic mice (Jackson laboratory) (Georgiades P, et al. VavCre transgenic mice: A tool for mutagenesis in hematopoietic and endothelial lineages. Genesis (New York, N.Y. : 2000). 2002;34(4):251-256. doi:l0.l002/gene.10161) to induce hematopoietic JAK2-V617F expression starting during mouse embryo genesis. To rule out any secondary effects of Cre recombinase JAK2+/+ (Cre+) and JAK2+/VF (Cre+) mice were VavCre-positive. 6.2 GTPase assays

Rapl activation was determined using pull-down assays with commercially available kits (Active Rapl Pull-Down and Detection Kit; Thermo Scientific) according to manufacturer’s instructions. 6.3 Isolation of granulocytes

Bone marrow was isolated from JAK2+/VF or JAK2+/+ mice following standard procedures. Isolation of granulocytes was performed as described previously (Hasenberg M, et al. Rapid immunomagnetic negative enrichment of neutrophil granulocytes from murine bone marrow for functional studies in vitro and in vivo. PloS one. 201 l;6(2):el73l4. doi:l0.l37l/joumal.pone.00l73l4) with minor modifications. Lineage-depletion was performed using Streptavidin Particles. The purity was determined by fluorescent staining with Gr-l and F4/80 antibodies.

6.4 Integrin activation assay

Integrin activation assay was performed, as previously described, with minor changes (Rehberg K, et al. The serine/threonine kinase Ndr2 controls integrin trafficking and integrin-dependent neurite growth. The Journal of neuroscience : the official journal of the Society for Neuroscience. 20l4;34(l5):5342-5354. doi:l0.l523/JNEUROSCI.2728- 13.2014). 1 x 106 murine granulocytes were resuspended in ice cold HBSS/l % FBS and incubated with 9EG7 antibody (CD29, clone 9EG7, BD Biosciences, #553715) for 60 min at 4°C. Cells were washed and incubated with secondary antibody coupled to R- phycoerythrin (1 :100) (clone Poly4054, Biolegend, #405406) for 30 min at 4°C. Binding 9EG7 was expressed as median fluorescence intensity, represented in the graphs in relation to total CD29 (clone HMbetal-l, Biolegend, #102215) expression.

6.5 Ligand binding assay 1 x 10⁶ murine granulocytes were incubated in the absence or presence of recombinant soluble VCAMl/Fc or ICAMl/Fc (10 pg/ml, R&D) for 60 min at 37°C. Cells were washed with ice cold HBSS/l % FBS followed by incubation with Allophycocyanin- conjugated AffiniPure F(ab’)2 fragment goat anti-human IgG, Fc-gamma fragment specific (1 :100, 30 min, 4°C) (Dianova, #109-136-098) and analyzed by flow cytometry. Binding of soluble ligand was expressed as median fluorescence intensity, calculated as fold of control to wild types. 6.6 Homing assay

Bone marrow was isolated from 10-12-week-old JAK2+/VF or JAK2+/+ mice as described before (Heidel FH, et al. The cell fate determinant Llgll influences HSC fitness and prognosis in AML. The Journal of experimental medicine. 2013;210(1): 15- 22. doi:l0.l084/jem.20l20596). 3 x 10⁶ whole bone marrow (WBM) cells of JAK2+/VF or JAK2+/+ mice (carrying the congenic marker CD45.2) were injected into CD45.1 (B6.SJL-Ptprca Pepcb/Boy) recipient mice. 16 h after injection recipient mice were sacrificed and spleens were evaluated. Cells were stained with respective antibodies and the amount of CD45.2-positive/l0⁶ cells in the myeloid compartment (monocytes Grl+ and F/4/80+, granulocytes Grl+) of the spleen was determined. Prior to homing assay, WBM cells from JAK+/VF mice were incubated with Rapl inhibitor GGTI-2147 (10 mM) for 30 min at 37°C or beta2 integrin blocking antibody (10 pg/ml, CD29, clone GAME-46, BD Bioscience, #555280) followed by subsequent injection into CD45.1 recipient mice as indicated.

6.7 Adhesion assay Adhesion assay was performed as described before (Kliche S, et al. CCR7-mediated LFA-l functions in T cells are regulated by 2 independent ADAP/SKAP55 modules. Blood. 20l2;l 19(3):777-785. doiTO.l l82/blood-20l 1-06-362269). Black 96 well microplates were precoated with recombinant Fc-free murine VCAM1 and ICAM1 (Leinco Technologies) (3 pg/ml) or BSA. Granulocytes were incubated with Rapl inhibitor GGTI-2147 (10 mM, 30 min) at 37°C. Cells were labeled with Calcein AM (2 pmol/l) and allowed to adhere for 30 min at 37°C. Fluorescence of the samples was monitored during washing steps using a Synergy HT plate reader (BioTek). 6.8 Partial ligation of the inferior vena cava (IVC)

10-12-week-old JAK2+/+ or JAK2+/V617F mice were anesthetized with sodium ketamine (100 mg/kg body weight, i.p.) and xylazine (10 mg/kg body weight, i.p.) and placed on a 37°C thermostatically controlled operating platform. After laparotomy, sterile saline was applied to the exteriorized intestines to prevent drying. The inferior vena cava (IVC) was gently separated from the aorta and partial ligation (stenosis) was induced by carefully placing a 30-gauge needle on the vessel and closure using a 7.0 polypropylene suture followed by immediate removal of the needle. All visible side branches were ligated as well. After surgery, peritoneum and skin were closed with continuous 4-0 polypropylene sutures. All animals received subcutaneous analgesic (buprenorphine 0.1 mg/kg) post-surgery. 4 h after partial ligation of the IVC, thrombi developed below the suture were analyzed for size and weight. For blockade of integrins a mixture of 200 pg VLA-4 antibody (Holzel Diagnostica) and 200 pg GAME46 antibody (BD Bioscience) were injected i.v. 30 min before inducing partial IVC ligation.

Claims

1. A pharmaceutical composition for use in therapy of JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) and of disorders or syndromes associated with JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN), the disorder or syndrome being selected from JAK2 V6l7F-associated splenomegaly, JAK2 V617F- associated increased risk of thrombosis and JAK2 V6l7F-associated inflammatory response syndrome, in an animal or human individual, the pharmaceutical composition comprising an integrin inhibitor, selected from an inhibitor of betal-integrin and an inhibitor of beta2 -integrin; wherein the inhibitor of betal-integrin is selected from monoclonal antibodies or ligands, specifically binding to the betal- or alpha 4-subunit of betal-integrin; and wherein the inhibitor of beta2 -integrin is selected from monoclonal antibodies or ligands, specifically binding to the beta2-subunit of beta2 -integrin.

2. The composition of claim 1, comprising an inhibitor of betal -subunit of betal - integrin.

3. The composition of claim 1 or 2, comprising an inhibitor of beta2-subunit of beta2 -integrin.

4. The composition of any one of claim 1 to 3, comprising an inhibitor of alpha4- subunit of betal-integrin.

5. The composition of any one of claim 1 to 3, comprising an inhibitor of alpha-L- subunit of beta2 -integrin.

6. The composition of any one of the preceding claims, further comprising an inhibitor of Rap 1 -GTPase.

7. The composition of any one of the preceding claims, wherein the JAK2 V617F- associated chronic myeloproliferative neoplasia (CMN) is essential thrombocythemia (ET), polycythemia vera (PV) or primary myelofibrosis (PMF).

8. An in vitro method for inhibiting adhesion of granulocytes, the method comprising the step of: contacting granulocytes with the composition of any one of claims 1 to 7.

9. An in vitro method for treating isolated tissue or cells having symptoms of JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN), the method comprising the step of: contacting the isolated tissue or cells with the composition of any one of claims 1 to 7.

10. The method of claim 9, wherein the isolated cells are selected from JAK-V617F- cells.

11. The method of claim 9 or 10, wherein the isolated cells are leukocytes, selected from monocytes and granulocytes.

12. A method of treatment of JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN) or a disorder or syndrome associated with JAK2 V6l7F-associated chronic myeloproliferative neoplasia (CMN), the disorder or syndrome being selected from JAK2 V6l7F-associated splenomegaly, JAK2 V6l7F-associated increased risk of thrombosis and JAK2 V6l7F-associated inflammatory response syndrome, the method comprising: administering a pharmaceutical composition comprising an integrin inhibitor, selected from: an inhibitor of beta 1 -integrin and an inhibitor of beta2-integrin, in a pharmaceutically acceptable carrier, to an animal or human individual in need thereof; wherein the inhibitor of beta 1 -integrin is selected from monoclonal antibodies or ligands, specifically binding to the betal- or alpha 4-subunit of beta 1 -integrin, and wherein the inhibitor of beta2 -integrin is selected from monoclonal antibodies or ligands, specifically binding to the beta2-subunit of beta2 -integrin.