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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.

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.

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.

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.

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.

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

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.

Human stem cell leukemia-lymphoma syndrome usually presents as a myeloproliferative disease (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-FGFR1 fusion tyrosine kinase. Current empirically-derived cytotoxic chemotherapy is inadequate 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 PLCγ, STAT5 and PI3K/AKT, required the proline-rich, but not the zinc-finger 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 myeloproliferative disease. 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 disease with organomegaly. Therefore, PKC412 may be a useful therapy for treatment of human stem cell leukemia-lymphoma syndrome.