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Purpose:The Philadelphia chromosome–negative myeloproliferative neoplasms (MPN) polycythemia vera, essential thrombocythemia, and primary myelofibrosis are characterized by JAK/STAT pathway activation. JAK inhibitors are approved for MPN treatment, but persistence has been observed, due to JAK/STAT reactivation.Experimental Design:Using MPN patient samples, JAK2-mutated cell lines, and MPN mouse models, we examined both the efficacy and mechanism by which crizotinib, the ALK/MET/RON/ROS1 inhibitor approved for the treatment of non–small cell lung cancer, alters MPN cell proliferation and JAK/STAT activation.Results:We found that crizotinib suppresses proliferation and activation of JAK/STAT signaling, and decreases the disease burden in the JAK2V617F mouse model of MPN. Furthermore, we found that crizotinib could overcome JAK inhibitor persistence to ruxolitinib. Interestingly, phosphorylation of the crizotinib target RON kinase was enhanced in ruxolitinib-persistent cells. We show that phospho-JAK2 and phospho-RON can physically interact to sustain JAK/STAT signaling, and that the combination of crizotinib and ruxolitinib disrupts this interaction. Furthermore, RON knockdown suppresses proliferation and activation of JAK/STAT signaling in JAK2-mutated cells, and RON deletion in a JAK2V617F mouse MPN model decreases the disease burden. We also observed RON hyperactivation in MPN patient cells, suggesting that RON may be an important target of crizotinib in MPN.Conclusions:In summary, we demonstrate that crizotinib has preclinical efficacy in MPN patient cells, JAK2-mutated cell lines, and a JAK2-mutated mouse model, and that the combination of crizotinib with JAK inhibitors suppresses JAK inhibitor persistence. Our work suggests that crizotinib should be investigated for the treatment of patients with MPN.

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Purpose:The Philadelphia chromosome–negative myeloproliferative neoplasms (MPN) polycythemia vera, essential thrombocythemia, and primary myelofibrosis are characterized by JAK/STAT pathway activation. JAK inhibitors are approved for MPN treatment, but persistence has been observed, due to JAK/STAT reactivation.Experimental Design:Using MPN patient samples, JAK2-mutated cell lines, and MPN mouse models, we examined both the efficacy and mechanism by which crizotinib, the ALK/MET/RON/ROS1 inhibitor approved for the treatment of non–small cell lung cancer, alters MPN cell proliferation and JAK/STAT activation.Results:We found that crizotinib suppresses proliferation and activation of JAK/STAT signaling, and decreases the disease burden in the JAK2V617F mouse model of MPN. Furthermore, we found that crizotinib could overcome JAK inhibitor persistence to ruxolitinib. Interestingly, phosphorylation of the crizotinib target RON kinase was enhanced in ruxolitinib-persistent cells. We show that phospho-JAK2 and phospho-RON can physically interact to sustain JAK/STAT signaling, and that the combination of crizotinib and ruxolitinib disrupts this interaction. Furthermore, RON knockdown suppresses proliferation and activation of JAK/STAT signaling in JAK2-mutated cells, and RON deletion in a JAK2V617F mouse MPN model decreases the disease burden. We also observed RON hyperactivation in MPN patient cells, suggesting that RON may be an important target of crizotinib in MPN.Conclusions:In summary, we demonstrate that crizotinib has preclinical efficacy in MPN patient cells, JAK2-mutated cell lines, and a JAK2-mutated mouse model, and that the combination of crizotinib with JAK inhibitors suppresses JAK inhibitor persistence. Our work suggests that crizotinib should be investigated for the treatment of patients with MPN.

Supplementary Figures and figure legends

Supplementary Methods, Figure and Table Legends 1-7 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

Supplementary Figure 1 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

PDF file - 190K, JAK2-inhibitor trial (proliferation assay, colony assay)

Supplementary Figure 3 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

PDF file - 626K, Leukemia phenotype (FACS, immunohistochemistry)

Supplementary Table 5 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

PDF file - 339K, LSC sorting strategy

PDF file - 91K

Supplementary Table 7 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

PDF file - 58K, Colony assay with and without cytokines

Supplementary Table 6 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

Supplementary Figure 4 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

Supplementary Figure 2 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineage compartments. Activation of JAK/STAT signaling is a major driver of all Ph-negative MPNs. During disease progression, MPN patients experience increased pro-inflammatory cytokine secretion, leading to remodeling of the bone marrow microenvironment and subsequent fibrosis. The JAK inhibitor ruxolitinib is an approved targeted therapy for MPN patients and has shown promise in its ability to reduce splenomegaly and the cytokine storm observed in patients. However, JAK inhibitors alone are not sufficient to reduce bone marrow fibrosis or to eliminate the JAK2-mutated clone. Furthermore, JAK inhibitor persistence, or reactivation of JAK/STAT signaling upon chronic JAK inhibitor treatment, has been observed in both MPN mouse models and MPN patients. Therefore, there is an urgent need for new treatment options in MPN. The tyrosine kinase RON, a member of the MET kinase family, has well-characterized roles in erythroblast proliferation and pro-inflammatory cytokine production. RON can be phosphorylated by JAK2 to stimulate erythroblast proliferation. However, the role of RON in MPN pathogenesis is unknown. We found that the ALK/MET/RON/ROS1 inhibitor crizotinib inhibited colony formation by MPN patient CD34+ cells, regardless of their disease subtype, mutation status, or JAK2 inhibitor treatment history (Figure 1A). To determine whether this is due to inhibition of the JAK/STAT signaling pathway, we performed phospho-flow cytometry of STAT3 and STAT5 in myelofibrosis patient erythroblasts treated with crizotinib ex vivo as well as Western blot analysis in the JAK2-mutated cell lines SET2 and HEL. We found that crizotinib inhibits the phosphorylation of JAK2, STAT3, and STAT5 (Figure 1B). Since crizotinib has not been reported to directly inhibit JAK2, we asked whether these effects of crizotinib in MPN cells could be explained by RON inhibition. Consistent with this hypothesis, we observed that shRNA knockdown of multiple RON isoforms also decreases the phosphorylation of JAK2, STAT5, and STAT3 in HEL cells (Figure 1C-D). To determine whether crizotinib can alter the MPN disease course in vivo, we tested crizotinib by oral gavage in the MPLW515L bone marrow transplant murine model of myelofibrosis at 100mg/kg daily for 2 weeks. We showed that crizotinib decreased the disease burden of MPL-W515L mice, as evidenced by decreased spleen and liver weights (Figure 1E). To determine the effects of RON genetic deletion on MPN pathogenesis, we tested whether genetic deletion of Stk (mouse gene for RON) impairs disease progression in the JAK2V617F bone marrow transplant MPN model by transplanting Stk-/- c-Kit+ bone marrow cells transduced with the JAK2V617F-GFP retrovirus into lethally irradiated recipients. We observed a significant delay in disease onset in Stk-/- transplant recipients compared to WT controls (Figure 1F). However, we found that Stk-/- mice have normal numbers of hematopoietic stem and progenitor cells, and normal bone marrow myeloid colony forming capacity, suggesting that RON is a safe therapeutic target. To determine whether RON plays a role in the JAK inhibitor persistence phenotype, we generated persistent cells by treating SET2 cells with increasing doses of ruxolitinib over 8 weeks, and confirmed persistent proliferation and JAK/STAT activation. Interestingly, we found that RON phosphorylation is enhanced in JAK inhibitor persistent cells, and that dual inhibition of RON and JAK2 overcomes JAK inhibitor persistence in SET2 cells (Figure 1G-H), suggesting that RON may potentiate the JAK2 persistence phenotype in response to ruxolitinib. Importantly, we showed by immunoprecipitation that phospho-RON and phospho-JAK2 physically interact in JAK inhibitor persistent SET2 cells, and that this interaction is disrupted by crizotinib (Figure 1I). In summary, our data demonstrate that RON kinase is a novel mediator of JAK/STAT signaling in MPNs, and that it plays a particularly important role in JAK inhibitor persistence. Our work suggests that therapeutic strategies to inhibit RON, such as crizotinib, should be investigated in MPN patients. Figure 1 Figure 1. Disclosures Halmos: Guardant Health: Membership on an entity's Board of Directors or advisory committees; Apollomics: Membership on an entity's Board of Directors or advisory committees; TPT: Membership on an entity's Board of Directors or advisory committees; Eli-Lilly: Research Funding; Advaxis: Research Funding; Blueprint: Research Funding; Elevation: Research Funding; Mirati: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Gritsman: iOnctura: Research Funding.

The Class IA PI3 kinase isoforms (p110α, β, and δ) transduce many growth factor signals that are important for the proliferation, differentiation, and self-renewal of hematopoietic stem cells (HSCs). Mutations in growth factor receptors or RAS proteins are commonly observed in patients with acute myeloid leukemia (AML), leading to activation of the PI3K/AKT pathway. Therefore, PI3K inhibition is an attractive therapeutic strategy for a large subset of AML patients. We previously reported that p110α is dispensable for HSC function, suggesting that redundancy exists between the Class IA isoforms in HSCs (Gritsman et al., J Clin Invest 2014). However, we have identified a specific role for p110α in RAS-mutated myeloid leukemia. Furthermore, we found that the p110α-selective inhibitor BYL-719 can sensitize RAS-mutated leukemic cells to the MEK inhibitor MEK-162. While PI3K inhibitors have multiple clinical indications, including in hematologic malignancies, it is still unclear whether PI3K plays an important role in normal HSC function. Upon environmental stresses like chemotherapy or infection, quiescent HSCs must enter the cell cycle to re-establish homeostasis and promote emergency myelopoiesis. To test for redundancy between p110α and p110δ in HSCs, we generated p110α and p110δ double knockout (DKO) mice, with germline deletion of p110δ and conditional deletion of p110α. DKO mice have leukopenia, anemia, and decreased numbers of lymphoid-primed multipotent progenitors (LMPPs) in the bone marrow, though HSC numbers are unchanged. In competitive repopulation assays, B-cell maintenance was severely impaired, while the myeloid and T-cell lineages were relatively preserved. This suggests that HSC function is not impaired at steady state. We performed transcriptome analysis of DKO, p110δ, and p110α KO HSCs and LMPPs after bone marrow transplantation. Gene set enrichment analysis revealed downregulation of gene sets associated with cell cycle progression. Interestingly, we also observed negative enrichment of inflammatory response gene sets in both p110δ KO and DKO HSCs and LMPPs. To examine the roles of p110δ and α in the hematopoietic stress response, we injected p110δ, DKO, and WT mice with 5-fluorouracil. We observed significantly decreased survival of 5-FU-treated DKO mice due to impaired hematopoietic recovery, associated with the failure of HSCs to enter the cell cycle. Consistent with this finding, we also observed a decrease in myeloid reconstitution by DKO HSCs in secondary competitive transplantation, suggesting that p110α and p110δ play redundant roles in emergency myelopoiesis. We also observed defects in downstream signaling in DKO HSCs and progenitors in response to IL1β or TNFα stimulation. Our results suggest that p110α and δ act in a redundant fashion to transduce specific inflammatory signals in HSPCs in response to hematopoietic stress. Our results have important implications for the use of PI3K inhibitors in combination with chemotherapy and in other hematopoietic stress conditions. Citation Format: Shayda Hemmati, Taneisha Sinclair, Meng Tong, Boris Bartholdy, Rachel Okabe, Kristina Ames, Leanne Ostrodka, Tamanna Haque, Imit Kaur, Anupriya Agarwal, Jean Zhao, Thomas Roberts, Kira Gritsman. The PI3K isoforms in myeloid leukemia and hematopoietic stem cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA18.

Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease., Several studies have recently demonstrated the role of the MSI2 RNA binding protein in normal and malignant haematopoietc stem cells. In this study, the authors show that MSI2 is required for maintaining myelodysplastic syndrome stem cells in mice and that MSI2 expression predicts poor prognosis in patients affected by this disease.

The genes encoding RAS family members are frequently mutated in juvenile myelomonocytic leukemia (JMML) and acute myeloid leukemia (AML). RAS proteins are difficult to target pharmacologically; therefore, targeting the downstream PI3K and RAF/MEK/ERK pathways represents a promising approach to treat RAS-addicted tumors. The p110α isoform of PI3K (encoded by Pik3ca) is an essential effector of oncogenic KRAS in murine lung tumors, but it is unknown whether p110α contributes to leukemia. To specifically examine the role of p110α in murine hematopoiesis and in leukemia, we conditionally deleted p110α in HSCs using the Cre-loxP system. Postnatal deletion of p110α resulted in mild anemia without affecting HSC self-renewal; however, deletion of p110α in mice with KRASG12D-associated JMML markedly delayed their death. Furthermore, the p110α-selective inhibitor BYL719 inhibited growth factor-independent KRASG12D BM colony formation and sensitized cells to a low dose of the MEK inhibitor MEK162. Furthermore, combined inhibition of p110α and MEK effectively reduced proliferation of RAS-mutated AML cell lines and disease in an AML murine xenograft model. Together, our data indicate that RAS-mutated myeloid leukemias are dependent on the PI3K isoform p110α, and combined pharmacologic inhibition of p110α and MEK could be an effective therapeutic strategy for JMML and AML.

Hematopoietic stem cells (HSCs) are a rare population in the bone marrow (BM) that mostly exists in a quiescent state. Upon environmental stresses such as infection, inflammatory signals are released and induce HSCs to proliferate to quickly re-establish homeostasis and maintain the blood system. Many hematopoietic growth factors and chemokines signal through PI3K pathway. In hematopoietic cells, 3 Class IA PI3K isoforms are expressed (p110α, β, and δ), each encoded by a different gene. While p110α and p110β are ubiquitously expressed, p110δ is enriched in leukocytes. We previously showed that p110α is dispensable for HSC function (Gritsman et al, J Clin Invest 2014 124:1794-1809), suggesting redundancy between Class I PI3K isoforms in HSCs. To test for potential redundancy between p110α and p110δ in HSCs, we generated mice with conditional deletion of p110α and germline deletion of p110δ (DKO mice). DKO mice have anemia, leukopenia and decreased BM cellularity. While there is no change in HSC numbers, the number of lymphoid-primed multipotent progenitors (LMPPs) is significantly decreased. In the primary competitive repopulation assay, DKO BM cells fail to reconstitute the B cell lineage, while the myeloid and T cell lineages were relatively preserved. However, in a secondary competitive transplantation setting, we also observed a significant decrease in myeloid reconstitution, suggesting that p110α and p110δ play redundant roles in emergency myelopoiesis. To examine effects of p110α and p110δ deletion on gene expression, we performed microarray analysis of WT, DKO, p110δ-/-, and p110α-/- hematopoietic stem and progenitor cells (HSPCs) after bone marrow transplantation. Gene set enrichment analysis revealed negative enrichment of gene sets associated with inflammatory response pathways in both DKO HSCs and LMPPs. In DKO HSCs, we also observed negative enrichment of gene sets associated with cell cycle progression. To further examine the roles of p110δ and p110α in the hematopoietic stress response, we injected DKO, p110δ-/-, and WT; Mx1-Cre mice with 5-fluorouracil (5-FU). We observed decreased survival of 5-FU-treated DKO mice associated with impaired hematopoietic recovery, and with the failure of HSCs to enter the cell cycle. Given the important roles of inflammatory signaling pathways in HSC activation and emergency myelopoiesis, we examined the roles of p110α and p110δ in signal transduction in HSPCs in response to IL1β or TNFα. Our phospho-flow cytometry analysis revealed a decrease in p38-MAPK phosphorylation in both p110δ-/- and DKO HSPCs, both at baseline and after stimulation with either IL1β or TNFα. To confirm these results, we stimulated p110δ KO and DKO cKit-enriched bone marrow cells with IL1β or TNFα. We observed a significant decrease in both p38-MAPK phosphorylation and phosphorylation of Akt at Ser473 in DKO cells, but not in p110δ-/- cells, both at baseline and with IL1-β or TNF-α stimulation. This suggests that both p110α and p110δ are required for optimal transduction of IL1β or TNFα in hematopoietic progenitors. Surprisingly, we found that DKO HSPCs can enter the cell cycle normally upon in vivo stimulation with lipopolysaccharide (LPS), which simulates bacterial infection. Our results suggest that p110α and δ act in a redundant fashion to transduce specific inflammatory signals in HSPCs, such as IL1β and TNFα, in response to hematopoietic stress. Our findings have important implications for the use of PI3K inhibitors in combination with chemotherapy and in the setting of infection or inflammation. Disclosures No relevant conflicts of interest to declare.

SummaryWe report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN.

Human cancers, including acute myeloid leukemia (AML), commonly display constitutive phosphoinositide 3-kinase (PI3K) AKT signaling. However, the exact role of AKT activation in leukemia and its effects on hematopoietic stem cells (HSCs) are poorly understood. Several members of the PI3K pathway, phosphatase and tensin homolog (Pten), the forkhead box, subgroup O (FOXO) transcription factors, and TSC1, have demonstrated functions in normal and leukemic stem cells but are rarely mutated in leukemia. We developed an activated allele of AKT1 that models increased signaling in normal and leukemic stem cells. In our murine bone marrow transplantation model using a myristoylated AKT1 (myr-AKT), recipients develop myeloproliferative disease, T-cell lymphoma, or AML. Analysis of the HSCs in myr-AKT mice reveals transient expansion and increased cycling, associated with impaired engraftment. myr-AKT–expressing bone marrow cells are unable to form cobblestones in long-term cocultures. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) rescues cobblestone formation in myr-AKT–expressing bone marrow cells and increases the survival of myr-AKT mice. This study demonstrates that enhanced AKT activation is an important mechanism of transformation in AML and that HSCs are highly sensitive to excess AKT/mTOR signaling.

Acquired mutations in the FLT3 receptor tyrosine kinase are common in acute myeloid leukemia and result in constitutive activation. The most frequent mechanism of activation is disruption of the juxtamembrane autoregulatory domain by internal tandem duplications (ITDs). FLT3-ITDs confer factor-independent growth to hematopoietic cells and induce a myeloproliferative syndrome in murine bone marrow transplant models. We and others have observed that FLT3-ITD activates STAT5 and its downstream effectors, whereas ligand-stimulated wild-type FLT3 (FLT3WT) does not. In vitro mapping of tyrosine phosphorylation sites in FLT3-ITD identified 2 candidate STAT5 docking sites within the juxtamembrane domain that are disrupted by the ITD. Tyrosine to phenylalanine substitution of residues 589 and 591 in the context of the FLT3-ITD did not affect tyrosine kinase activity, but abrogated STAT5 activation. Furthermore, FLT3-ITD–Y589/591F was incapable of inducing a myeloproliferative phenotype when transduced into primary murine bone marrow cells, whereas FLT3-ITD induced myeloproliferative disease with a median latency of 50 days. Thus, the conformational change in the FLT3 juxtamembrane domain induced by the ITD activates the kinase through dysregulation of autoinhibition and results in qualitative differences in signal transduction through STAT5 that are essential for the transforming potential of FLT3-ITD in vivo.

AMN107 is a small molecule tyrosine kinase inhibitor developed, in the first instance, as a potent inhibitor of breakpoint cluster region-abelson (BCR-ABL). We tested its effectiveness against fusion tyrosine kinases TEL-platelet-derived growth factor receptorbeta (TEL-PDGFRbeta) and FIP1-like-1 (FIP1L1)-PDGFRalpha, which cause chronic myelomonocytic leukemia and hypereosinophilic syndrome, respectively. In vitro, AMN107 inhibited proliferation of Ba/F3 cells transformed by both TEL-PDGFRbeta and FIP1L1-PDGFRalpha with IC50 (inhibitory concentration 50%) values less than 25 nM and inhibited phosphorylation of the fusion kinases and their downstream signaling targets. The imatinib mesylate-resistant mutant TEL-PDGFRbeta T681I was sensitive to AMN107, whereas the analogous mutation in FIP1L1-PDGFRalpha, T674I, was resistant. In an in vivo bone marrow transplantation assay, AMN107 effectively treated myeloproliferative disease induced by TEL-PDGFRbeta and FIP1L1-PDGFRalpha, significantly increasing survival and disease latency and reducing disease severity as assessed by histopathology and flow cytometry. In summary, AMN107 can inhibit myeloid proliferation driven by TEL-PDGFRbeta and FIP1L1-PDGFRalpha and may be a useful drug for treatment of patients with myeloproliferative disease who harbor these kinase fusions.

Early placental insulin-like protein (INSL4 or EPIL) is a member of the insulin superfamily of hormones, which is highly expressed in the placenta. We have confirmed this at term and shown it to be expressed by the maternal decidua. Although an abundance of locally acting growth factors are produced within the uterus during pregnancy, we hypothesized that INSL4 plays an important role in fetal and placental growth. We have demonstrated with cell lines and primary cells that it has a growth-inhibitory effect by causing apoptosis and loss of cell viability. We used primary amniotic epithelial cells for flow cytometry to show that INSL4 caused apoptosis, which was dose-related and significant (P < 0.05) at 50 ng/ml. This was confirmed by measurement of the nuclear matrix protein in the media. In comparison, relaxin treatment (up to 200 ng/ml) had no effect on apoptosis. The addition of INSL4 (3-30 ng/ml) also caused a loss of cell viability, although it had no effect on the numbers of cells at different phases of the cell cycle. Placental apoptosis is an important process in both normal placental development and in fetal growth restriction. Therefore, an in vivo clinical correlate was sought in fraternal twins exhibiting discordant growth. Expression of the INSL4 gene was doubled in the placenta of the growth-restricted twin compared to the normally grown sibling, suggesting that it may be linked to a higher level of apoptosis and loss of cell viability and, therefore, that it may contribute to fetal growth restriction.

Reccurent chromosomal translocation t(4;14) (p16.3;q32.3) occurs in patients with multiple myeloma (MM) and is associated with ectopic overexpression of fibroblast growth factor receptor 3 (FGFR3) that sometimes may contain the activation mutations such as K650E thanatophoric dysplasia type II (TDII). Although there have been significant advances in therapy for MM including the use of proteasome inhibitors, t(4;14) MM has a particularly poor prognosis and most patients still die from complications related to their disease or therapy. One potential therapeutic strategy is to inhibit FGFR3 in those myeloma patients that overexpress the receptor tyrosine kinase due to chromosomal translocation. Here we evaluated PKC412, a small molecule tyrosine kinase inhibitor, for treatment of FGFR3-induced hematopoietic malignancies. PKC412 inhibited kinase activation and proliferation of hematopoietic Ba/F3 cells transformed by FGFR3 TDII or a TEL-FGFR3 fusion. Similar results were obtained in PKC412 inhibition of several different t(4;14)-positive human MM cell lines. Furthermore, treatment with PKC412 resulted in a statistically significant prolongation of survival in murine bone marrow transplant models of FGFR3 TDII-induced pre-B cell lymphoma, or a peripheral T-cell lymphoma associated TEL-FGFR3 fusion-induced myeloproliferative disease. These data indicate that PKC412 may be a useful molecularly targeted therapy for MM associated with overexpression of FGFR3, and perhaps other diseases associated with dysregulation of FGFR3 or related mutants.

MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essential for MOZ-TIF2–induced leukemic transformation itself. Lack of STAT5 in fetal liver cells caused rapid differentiation and loss of replating capacity of MOZ-TIF2–transduced cells enriched for LSCs. Furthermore, mice serially transplanted with Stat5−/− MOZ-TIF2 leukemic cells develop AML with longer disease latency and finally incomplete penetrance when compared with mice transplanted with Stat5+/+ MOZ-TIF2 leukemic cells. These data suggest that STAT5AB is required for the self-renewal of LSCs and represents a combined signaling node of FLT3-ITD and MOZ-TIF2 driven leukemogenesis. Therefore, targeting aberrantly activated STAT5 or rewired downstream signaling pathways may be a promising therapeutic option. Cancer Res; 73(1); 373–84. ©2012 AACR.

Aging degrades hematopoietic stem cell (HSC) functions, including stress response; however, the involved molecular pathways are incompletely defined. Murine BM conditionally deleted for One-Twenty-Two-1 (Ott1), is able to maintain lifelong hematopoiesis and has preserved numbers of long-term HSCs, yet cannot repopulate nor sustain itself after transplantation against a competitor even when Ott1 is excised after engraftment. We show, specifically under replicative stress, that Ott1-deleted HSCs have a significant reduction of the G0 cell-cycle fraction associated with self-renewal and undergo early failure. Therefore, Ott1 is required to preserve HSC quiescence during stress but not steady-state hematopoiesis. Reduced tolerance of replicative stress, increased myeloid potential, and greater absolute numbers are mutual characteristics of both Ott1-deleted and aged HSCs, and comparison of their gene expression profiles reveals a shared signature. Ott1-deleted HSCs share multiple aging-associated physiologic changes, including increases in NF-κB activation and DNA damage. Loss of Ott1 causes increased reactive oxygen species; however, antioxidant treatment does not rescue the competitive defect, indicating the existence of additional essential Ott1-dependent HSC pathways. In conclusion, our data establish a requirement for Ott1 in stress hematopoiesis and suggest that Ott1-dependent processes may converge with those affected by aging.

About 10% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) that lack mutations in JAK2 harbor an activating mutation in the thrombopoietin receptor, MPLW515L. Distinct from the JAK2V617F retroviral transplant model, the MPLW515L model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis. We have tested EXEL-8232, an experimental potent JAK2 inhibitor, for efficacy in suppression of thrombocytosis in vivo and for its ability to attenuate MPLW515L myeloproliferative disease. EXEL-8232 was administered for 28 days q12 h by oral gavage at doses of 30 or 100 mg/kg, prospectively. Animals treated with EXEL-8232 at 100 mg/kg had normalized high platelet counts, eliminated extramedullary hematopoiesis in the spleen and eliminated bone marrow fibrosis, whereas the wild-type controls did not develop thrombocytopenia. Consistent with a clinical response in this model, we validated surrogate end points for response to treatment, including a reduction of endogenous colony growth and signaling inhibition in immature erythroid and myeloid primary cells both in vitro and upon treatment in vivo. We conclude that EXEL-8232 has efficacy in treatment of thrombocytosis in vivo in a murine model of ET and MF, and may be of therapeutic benefit for patients with MPL-mutant MPN.

Fusion oncogenes in acute myeloid leukemia (AML) promote self-renewal from committed progenitors, thereby linking transformation and self-renewal pathways. Like most cancers, AML is a genetically and biologically heterogeneous disease, but it is unclear whether transformation results from common or overlapping genetic programs acting downstream of multiple mutations or by the engagement of unique genetic programs acting cooperatively downstream of individual mutations. This distinction is important, because the involvement of common programs would imply the existence of common molecular targets to treat AML, no matter which oncogenes are involved. Here we show that the ability to promote self-renewal is a generalized property of leukemia-associated oncogenes. Disparate oncogenes initiated overlapping transformation and self-renewal gene expression programs, the common elements of which were defined in established leukemic stem cells from an animal model as well as from a large cohort of patients with differing AML subtypes, where they strongly predicted pathobiological character. Notably, individual genes commonly activated in these programs could partially phenocopy the self-renewal function of leukemia-associated oncogenes in committed murine progenitors. Furthermore, they could generate AML following expression in murine bone marrow. In summary, our findings reveal the operation of common programs of self-renewal and transformation downstream of leukemia-associated oncogenes, suggesting that mechanistically common therapeutic approaches to AML are likely to be possible, regardless of the identity of the driver oncogene involved. Cancer Res; 71(12); 4117–29. ©2011 AACR.

available in PMC 2011 May 10., RNA-binding proteins of the Musashi (Msi) family are expressed in stem cell compartments and in aggressive tumors, but they have not yet been widely explored in the blood. Here we demonstrate that Msi2 is the predominant form expressed in hematopoietic stem cells (HSCs), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of human MSI2 in a mouse model increases HSC cell cycle progression and cooperates with the chronic myeloid leukemia–associated BCR-ABL1 oncoprotein to induce an aggressive leukemia. MSI2 is overexpressed in human myeloid leukemia cell lines, and its depletion leads to decreased proliferation and increased apoptosis. Expression levels in human myeloid leukemia directly correlate with decreased survival in patients with the disease, thereby defining MSI2 expression as a new prognostic marker and as a new target for therapy in acute myeloid leukemia (AML)., Leukemia & Lymphoma Society of America, National Institutes of Health (U.S.) (Ruth L. Kirschstein Fellowship), National Institute of Diabetes and Digestive and Kidney Diseases (U.S.), National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) ( Kidney Diseases Career Development Award)

We report that TG101348, a selective small-molecule inhibitor of JAK2 with an in vitro IC50 of approximately 3 nM, shows therapeutic efficacy in a murine model of myeloproliferative disease induced by the JAK2V617F mutation. In treated animals, there was a statistically significant reduction in hematocrit and leukocyte count, a dose-dependent reduction/elimination of extramedullary hematopoiesis, and, at least in some instances, evidence for attenuation of myelofibrosis. There were no apparent toxicities and no effect on T cell number. In vivo responses were correlated with surrogate endpoints, including reduction/elimination of JAK2V617F disease burden assessed by quantitative genomic PCR, suppression of endogenous erythroid colony formation, and in vivo inhibition of JAK-STAT signal transduction as assessed by flow cytometric measurement of phosphorylated Stat5.

+ The first four authors contributed equally to this project. Myelodysplastic syndromes (MDS) are a group of blood cell disorders, characterized by ineffective hematopoiesis and severe cytopenias, which often transform to acute leukemia. MDS is also considered to be a clonal stem cell disease driven by alterations that are both genetic and epigenetic. However, it remains unclear how stem cell function is dysregulated and what factors drive these alterations in MDS HSCs. MSI2 is an important RNA-binding protein in normal HSC maintenance and can promote aggressive myeloid leukemia. Our preliminary data indicate that MSI2 expression is increased in high-risk MDS compared to low-risk MDS and correlates with poor survival. In order to model the role of MSI2 in MDS, we utilized the NUP98-HOXD13 transgenic (NHD13) model, which recapitulates many salient features of MDS including, leukopenia, severe anemia, erythroid dysplasia and leukemic transformation. Despite the lethal MDS or AML disease found in primary NHD13 animals, bone marrow cells transplanted into congenic mice generate a non-lethal MDS that rarely transform. Depletion of Msi2 utilizing a conditional knockout (NHD13-Msi2f/f -MX1-Cre) reversed the MDS phenotype and after one month the diseased HSPCs were eliminated. Conversely, we found that tetracycline inducible MSI2 overexpression in the context of the NHD13 transgene (NHD13/MSI2 mice) resulted in a worse MDS disease and a fully penetrant and lethal transformation to an AML, which was further accelerated during serial transplantation. AML arising in NHD13/MSI2 mice remained dependent on sustained MSI2 overexpression as mice removed from doxycycline demonstrated improved survival. Most interestingly, MSI2 overexpression expanded and maintained a more activated (G1) MDS hematopoietic stem and progenitor compartment (HSPC) in NHD13 cells. Gene expression profiling of the LSKs (Lineagelo, c-Kit+, Sca1+) before disease progression identified 891 significant genes, of which 137 genes were up-regulated (log2 fold change > 0) and 754 genes were down-regulated (log2 fold change Disclosures No relevant conflicts of interest to declare.

An acquired somatic mutation, Jak2V617F, was recently discovered in most patients with polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), and essential thrombocythemia (ET). To investigate the role of this mutation in vivo, we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F, but not Jak2wt, resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit, leukocytosis, megakaryocyte hyperplasia, extramedullary hematopoiesis resulting in splenomegaly, and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors, although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity, and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore, we observed strain-specific differences in phenotype, with Balb/c mice demonstrating markedly elevated leukocyte counts, splenomegaly, and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans.

Early placental insulin-like protein (INSL4 or EPIL) is a member of the insulin superfamily of hormones, which is highly expressed in the placenta. We have confirmed this at term and shown it to be expressed by the maternal decidua. Although an abundance of locally acting growth factors are produced within the uterus during pregnancy, we hypothesized that INSL4 plays an important role in fetal and placental growth. We have demonstrated with cell lines and primary cells that it has a growth-inhibitory effect by causing apoptosis and loss of cell viability. We used primary amniotic epithelial cells for flow cytometry to show that INSL4 caused apoptosis, which was dose-related and significant (P0.05) at 50 ng/ml. This was confirmed by measurement of the nuclear matrix protein in the media. In comparison, relaxin treatment (up to 200 ng/ml) had no effect on apoptosis. The addition of INSL4 (3-30 ng/ml) also caused a loss of cell viability, although it had no effect on the numbers of cells at different phases of the cell cycle. Placental apoptosis is an important process in both normal placental development and in fetal growth restriction. Therefore, an in vivo clinical correlate was sought in fraternal twins exhibiting discordant growth. Expression of the INSL4 gene was doubled in the placenta of the growth-restricted twin compared to the normally grown sibling, suggesting that it may be linked to a higher level of apoptosis and loss of cell viability and, therefore, that it may contribute to fetal growth restriction.

Human stem cell leukemia-lymphoma syndrome usually presents itself as a myeloproliferative disorder (MPD) that evolves to acute myeloid leukemia and/or lymphoma. The syndrome associated with t(8;13)(p11;q12) results in expression of the ZNF198-fibroblast growth factor receptor (FGFR) 1 fusion tyrosine kinase. Current empirically derived cytotoxic chemotherapy is inadequate for treatment of this disease. We hypothesized that small-molecule inhibitors of the ZNF198-FGFR1 fusion would have therapeutic efficacy. We characterized the transforming activity of ZNF198-FGFR1 in hematopoietic cells in vitro and in vivo . Expression of ZNF198-FGFR1 in primary murine hematopoietic cells caused a myeloproliferative syndrome in mice that recapitulated the human MPD phenotype. Transformation in these assays, and activation of the downstream effector molecules PLC-γ, STAT5, and phosphatidylinositol 3-kinase/AKT, required the proline-rich domains, but not the ZNF domains, of ZNF198. A small-molecule tyrosine kinase inhibitor, PKC412 ( N -benzoyl-staurosporine) effectively inhibited ZNF198-FGFR1 tyrosine kinase activity and activation of downstream effector pathways, and inhibited proliferation of ZNF198-FGFR1 transformed Ba/F3 cells. Furthermore, treatment with PKC412 resulted in statistically significant prolongation of survival in the murine model of ZNF198-FGFR1-induced MPD. Based in part on these data, PKC412 was administered to a patient with t(8;13)(p11;q12) and was efficacious in treatment of progressive myeloproliferative disorder with organomegaly. Therefore, PKC412 may be a useful therapy for treatment of human stem cell leukemia-lymphoma syndrome.

NO synthase 2 (NOS2) plays an important role in endotoxemia through overproduction of NO. Distamycin A (Dist A) belongs to a class of drugs termed minor-groove DNA binders, which can inhibit transcription factor binding to AT-rich regions of DNA. We and others have previously shown that AT-rich regions of DNA surrounding transcription factor binding sites in the NOS2 promoter are critical for NOS2 induction by inflammatory stimuli in vitro. Therefore, we hypothesized that Dist A would attenuate NOS2 up-regulation in vivo during endotoxemia and improve animal survival. C57BL/6 wild-type (WT) mice treated with Dist A and LPS (endotoxin) showed significantly improved survival compared with animals treated with LPS alone. In contrast, LPS-treated C57BL/6 NOS2-deficient (NOS2−/−) mice did not benefit from the protective effect of Dist A on mortality from endotoxemia. Treatment with Dist A resulted in protection from hypotension in LPS-treated WT mice, but not in NOS2−/− mice. Furthermore, LPS-induced NOS2 expression was attenuated in vivo (WT murine tissues) and in vitro (primary peritoneal and RAW 264.7 murine macrophages) with addition of Dist A. Dist A selectively decreased IFN regulatory factor-1 DNA binding in the enhancer region of the NOS2 promoter, and this IFN regulatory factor-1 site is critical for the effect of Dist A in attenuating LPS induction of NOS2. Our data point to a novel approach in modulating NOS2 expression in vivo during endotoxemia and suggest the potential for alternative treatment approaches for critical illness.

Abstract 3620 Poster Board III-556 PIK3CA, which encodes the p110α catalytic isoform of PI3 kinase (PI3K), is mutated in many human cancers, and is an attractive therapeutic target. However, PI3K may also be important during hematopoiesis, as it is activated by hematopoietic growth factor receptors which control hematopoietic stem cell (HSC) and progenitor proliferation, differentiation, and self-renewal, such as erythropoietin receptor (epoR), c-kit receptor, and fms-like tyrosine kinase 3 (FLT3). In hematopoietic cells, receptor tyrosine kinases signal through the catalytic p110 subunit of PI3K, which has 3 isoforms (α, β, δ). However, the roles of PI3K and its specific catalytic isoforms in normal HSC function are poorly understood. We hypothesized that signaling through the p110α isoform is important for hematopoiesis and HSC self-renewal. We have used the Cre-loxP system to delete p110α in the HSCs of adult mice by breeding p110αF/F mice to Mx1-Cre transgenic mice. p110αF/F;Mx1-Cre+ (Cre+) mice and their p110αF/F (Cre-) littermates were injected with PolyI-PolyC (pIpC) at 4-6 weeks of age to induce Cre-mediated excision at the PIK3CA locus specifically in hematopoietic cells. Deletion of p110α in the bone marrow (BM) was verified by PCR and by immunoblot. We observed that, by four weeks after pIpC treatment, Cre+ mice developed microcytic anemia compared with Cre- littermates, characterized by a decreased mean hemoglobin (p Disclosures: Gilliland: Merck Research Laboratories: Employment. Roberts:Novartis Pharmaceuticals, Inc.: Consultancy. Zhao:Novartis Pharmaceuticals, Inc.: Consultancy.

Approximately 50% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) lack activating mutations in JAK2. Among these patients, ~10% harbor an activating mutation in the thrombopoietin receptor, MPLW515L. We have reported that expression of MPLW515L in a murine bone marrow transplant model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis, that are not observed in the JAK2V617F model. These observations provide an opportunity to assess the efficacy of small molecule JAK2 inhibitors on a myeloproliferative disease (MPD) induced by MPLW515L in vivo, and to determine whether such inhibitors attenuate thrombocytosis. We have tested EXEL-8232 for efficacy in suppression of thrombocytosis in vivo, and for its ability to attenuate JAK2V617F-negative MPD mediated by MPLW515L. EXEL-8232 is a potent small molecule inhibitor of JAK2 and is structurally similar to XL019, a compound currently in clinical trials for MF and polycythemia vera. EXEL-8232 is selective for JAK2 with a biochemical IC50 of 2 nM, and abolished constitutive phosphorylation of JAK2 and STAT5, as well as cytokine-independent growth, of Ba/F3 cells in vitro. After disease was established 12 days post-bone marrow transplantation, EXEL-8232 was administered for 28 days q12h by oral gavage at doses of 30mg/kg or 100mg/kg respectively. Animals treated with 100mg/kg normalized high platelet counts in excess of2 million/ml and normalized leukocytosis from a median of 134,000/ml in vehicle treated controls. Furthermore, drug treatment eliminated extramedullary hematopoiesis in the spleen, as well as bone marrow fibrosis. Of note, EXEL-8232 had no impact on erythrocytosis in diseased animals or in wild type controls, and wild type animals treated with either dosage of 30mg/kg or 100mg/kg did not develop thrombocytopenia. Consistent with these clinical responses, the surrogate endpoints for response to treatment included a reduction of genomic disease burden in the 100mg/kg treated arm (p

Self-renewal is a prerequisite for cancer initiation and maintenance, however the origins and molecular basis of self-renewal in malignant cells remain elusive. Recently, we and others have shown that certain leukemia-associated fusion oncogenes (such as MOZ-TIF2 and MLL fusions) can alter self-renewal in, and generate acute myeloid leukemia (AML) from, committed murine myeloid progenitor cells. AML is a highly heterogeneous disease, both genetically and biologically, and is associated with many differing combinations of mutations and a variable clinical course. However, it is not currently known whether an alteration of self-renewal is a more widespread finding in AML. Moreover, it is also not known whether alterations of self-renewal are mediated by common genetic programs downstream of multiple individual mutations or through the engagement of unique programs downstream of individual mutations. This distinction is important, as the demonstration of common pathways may identify common critical molecular targets for the treatment of AML. We now demonstrate, using retroviral expression of the AML1-ETO (RUNX1-RUNX1T1) and NUP98-HOXA9 fusion proteins in committed myeloid progenitors, that the ability to alter self-renewal is a more generalized property of leukemia-associated transcription factor fusion oncogenes. Furthermore, to investigate the molecular basis for the reestablishment of self-renewal we compare expression differences between granulocyte monocyte progenitors (GMP) directly transduced with MOZ-TIF2, AML1-ETO and NUP98-HOXA9 retroviruses and GMP transduced with empty vector retrovirus, to identify common candidate genes and molecular pathways involved in self-renewal downstream of these fusion oncogenes. Thereafter, we validate the expression of these early genetic programs in functionally defined murine leukemia stem cells and bulk human leukemias and perform functional experiments to validate the effects of individual candidate genes within the programs on self-renewal and transformation. Our data demonstrate AML1-ETO and NUP98-HOXA9 alter self-renewal in and NUP98-HOXA9 generates AML from committed myeloid progenitors. Disparate leukemia-associated oncogenes (MOZ-TIF2, AML1-ETO and NUP98-HOXA9) initiate an early, common self-renewal signature of 167 genes (FDR level p

Hedgehog (Hh) pathway proteins are a highly conserved family of intracellular signaling molecules that are critical for the development of multiple organs and tissues, and play a role in cell fate determination of self-renewing tissues in the adult. Mutations that impair Hh signaling have been associated with developmental abnormalities, and recent studies indicate that Hh plays an important role in hemangioblast formation and in adult hematopoiesis, as well as in the differentiation and proliferation of hematopoietic stem cells (HSC) and progenitor cells. We used a genetic and pharmacologic approach to define the role of the Hh pathway in adult hematopoiesis and leukemogenesis. We report the unexpected finding that loss of Hh signaling through conditional deletion of Smoothened (Smo) in the adult hematopoietic compartment has no effect on adult hematopoiesis, including peripheral blood count, number or cell cycle status of stem and progenitor cells, hematopoietic colony forming potential, long-term repopulating activity in competitive repopulation assays, or stress-response to serial 5-fluorouracil treatment. In support of these observations based on genetic inactivation of the pathway, we observed that pharmacologic inhibition of Hh signaling with a potent and highly selective small molecule antagonist of Smo has no apparent effect on hematopoiesis in the mouse in vivo. In addition, we observed that Hh signaling is not required for the development of MLL-AF9 mediated leukemia. Taken together, these data indicate that Hh signaling is dispensable for normal hematopoietic development and leukemogenesis, and that pharmacologic inhibition of Hh signaling, as a therapeutic strategy in treatment of solid tumors with constitutive Hh pathway activation is not likely to be associated with unmanageable hematopoietic toxicity.

The JAK2V617F mutation is present in the majority of cases of myeloproliferative disease, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and is an attractive candidate for molecularly targeted therapy. However, the potential toxicities of JAK2 inhibition in vivo, and identification of appropriate surrogate endpoints for response, are challenges that may limit clinical usefulness in treatment of these relatively indolent diseases. We report efficacy and assessment of surrogate endpoints for response of a small molecule JAK2 inhibitor, TG101348 in a murine model of polycythemia vera. TG101348 is selective for JAK2 with an in vitro IC50 of ∼3 nM that is ∼334 fold more potent than for inhibition of JAK3. TG101348 showed therapeutic efficacy in the murine model of PV that included a statistically significant reduction in hematocrit, normalization of white blood cell count, a dose dependent reduction/elimination of extramedullary hematopoiesis in the spleen and liver, and marked attenuation of myelofibrosis. Consistent with its selective inhibition of JAK2 and not JAK3, there was no significant change in T-cell number in treated animals. These clinical responses correlated with surrogate endpoints for response, including reduction or elimination of JAK2V617F expressing clones based on quantitative genomic PCR, suppression of JAK2V617F positive endogenous erythroid colony growth of JAK2V617F MPD bone marrow, and inhibition of JAK-STAT signal transduction as assessed by phosphoflow cytometry for phosphorylated STAT5. Thus, TG101348 is efficacious in treatment of a murine model of PV, and surrogate endpoints have been identified that may be of value in clinical trials in humans.

MOZ-TIF2 is expressed as a consequence of the chromosomal inversion inv(8)(p11q13) and is associated with AML FAB subtypes M4 and M5. Mice transplanted with MOZ-TIF2 succumb to monoclonal or oligoclonal leukemias with a long median disease latency, indicating that cooperating mutations are required for MOZ-TIF2 mediated leukemogenesis. The presence of a FLT3-ITD mutation in a patient with inv(8)(p11q13) has previously been reported, suggesting FLT3-ITD as a candidate cooperating mutation. Here we report that FLT3-ITD functionally cooperates with MOZ-TIF2 in co-transduction experiments in both a serial replating and murine bone marrow transplantation assay to induce AML that is transplantable to secondary recipients. At limit dilution, both MOZ-TIF2 and FLT3-ITD retroviruses are present, demonstrating cooperative effect. Moreover, tyrosine to phenylalanine mutations of FLT3-ITD residues 589 and 591, which we have previously reported to abrogate Stat5 signaling, also abolish the ability of FLT3-ITD to cooperate with MOZ-TIF2 both in vivo and in vitro. Furthermore, a constitutively active Stat5 mutant supports factor independent serial replating activity of MOZ-TIF2 in vitro. These data suggest a multi-step transformation model in which constitutive downstream Stat5 signaling by FLT3-ITD cooperates with MOZ-TIF2 in AML induction, and indicate that FLT3-ITD potentiates the properties of self-renewal in hematopoietic progenitors through activation of STAT5.

Leukemias are often associated with aberrant tyrosine kinase activity that occurs as a result of chromosomal translocations. These mutations are able to confer a proliferative and survival advantage to leukemic cells, as well as cooperate with other mutations that impair cell differentiation, thus leading to the development of leukemia. NUP214-ABL1 is one such recently identified fusion gene that is generated by episomal amplification. The presence of the fusion was recently identified in approximately 6% of patients with T-cell acute lymphoblastic leukemia (T-ALL). By the use of a murine retroviral bone marrow transplantation model we have demonstrated that mice transplanted with NUP214-ABL1 transduced bone marrow cells developed either a myeloproliferative disorder (MPD) with a disease latency of 70 to 118 days or a T cell lymphoblastic leukemia with a disease latency of 115 to 124 days. The myeloproliferative phenotype was characterized by splenomagaly and leukocytosis, and analysis of the histopathology revealed extramedullary hematopoiesis in the liver, lung, kidney and Peyer’s patches, and an increase of peripheral blood neutrophils. Flow cytometry of single cell suspensions from spleen and bone marrow samples of mice with a myeloproliferative phenotype demonstrated an increase of Gr-1+/Mac-1+ cells (approximately 70%). Two of the mice that were transplanted with NUP214-ABL1 transduced bone marrow cells developed T cell lymphomas that were characterized by large thymomas, a phenotype that is consistent with other models of activated tyrosine kinases over long disease latencies. Histopathological analysis of the thymi revealed effacement of normal thymic architecture as well as T cell infiltrate into the surrounding skeletal muscle. In addition, flow cytometric analysis revealed a significant increase in the CD4+/CD8+ T cell population in the thymi of these animals. No disease was observed in secondary transplant recipients following 60 days of observation. In conclusion, these results indicate that NUP214-ABL1 is able to cause either a myeloproliferative disease or a T cell lymphoma over longer latencies in mice, the latter being similar to the phenotype observed in humans with expression of the NUP214-ABL1 fusion. These findings provide a useful model for future experiments to determine if there is a contribution of other mutations together with the NUP214-ABL1 fusion towards the development of a T-ALL phenotype in mice.