Regulation of HOX gene expression in AML.

As key developmental regulators, HOX cluster genes have varied and context-specific roles in normal and malignant hematopoiesis. A complex interaction of transcription factors, epigenetic regulators, long non-coding RNAs and chromatin structural changes orchestrate HOX expression in leukemia cells. In this review we summarize molecular mechanisms underlying HOX regulation in clinical subsets of AML, with a focus on NPM1 mutated (NPM1mut) AML comprising a third of all AML patients. While the leukemia initiating function of the NPM1 mutation is clearly dependent on HOX activity, the favorable treatment responses in these patients with upregulation of HOX cluster genes is a poorly understood paradoxical observation. Recent data confirm FOXM1 as a suppressor of HOX activity and a well-known binding partner of NPM suggesting that FOXM1 inactivation may mediate the effect of cytoplasmic NPM on HOX upregulation. Conversely the residual nuclear fraction of mutant NPM has also been recently shown to have chromatin modifying effects permissive to HOX expression. Recent identification of the menin-MLL interaction as a critical vulnerability of HOX-dependent AML has fueled the development of menin inhibitors that are clinically active in NPM1 and MLL rearranged AML despite inconsistent suppression of the HOX locus. Insights into context-specific regulation of HOX in AML may provide a solid foundation for targeting this common vulnerability across several major AML subtypes.

PRL2 Phosphatase Promotes Oncogenic KIT Signaling in Leukemia Cells through Modulating CBL Phosphorylation.

Receptor tyrosine kinase KIT is frequently activated in acute myeloid leukemia (AML). While high PRL2 (PTP4A2) expression is correlated with activation of SCF/KIT signaling in AML, the underlying mechanisms are not fully understood. We discovered that inhibition of PRL2 significantly reduces the burden of oncogenic KIT-driven leukemia and extends leukemic mice survival. PRL2 enhances oncogenic KIT signaling in leukemia cells, promoting their proliferation and survival. We found that PRL2 dephosphorylates CBL at tyrosine 371 and inhibits its activity toward KIT, leading to decreased KIT ubiquitination and enhanced AKT and ERK signaling in leukemia cells. IMPLICATIONS: Our studies uncover a novel mechanism that fine-tunes oncogenic KIT signaling in leukemia cells and will likely identify PRL2 as a novel therapeutic target in AML with KIT mutations.

Nore1 inhibits age-associated myeloid lineage skewing and clonal hematopoiesis but facilitates termination of emergency (stress) granulopoiesis.

Age-associated bone marrow changes include myeloid skewing and mutations that lead to clonal hematopoiesis. Molecular mechanisms for these events are ill defined, but decreased expression of Irf8/Icsbp (interferon regulatory factor 8/interferon consensus sequence binding protein) in aging hematopoietic stem cells may contribute. Irf8 functions as a leukemia suppressor for chronic myeloid leukemia, and young Irf8-/- mice have neutrophilia with progression to acute myeloid leukemia (AML) with aging. Irf8 is also required to terminate emergency granulopoiesis during the innate immune response, suggesting this may be the physiologic counterpart to leukemia suppression by this transcription factor. Identifying Irf8 effectors may define mediators of both events and thus contributors to age-related bone marrow disorders. In this study, we identified RASSF5 (encoding Nore1) as an Irf8 target gene and investigated the role of Nore1 in hematopoiesis. We found Irf8 activates RASSF5 transcription and increases Nore1a expression during emergency granulopoiesis. Similar to Irf8-/- mice, we found that young Rassf5-/- mice had increased neutrophils and progressed to AML with aging. We identified enhanced DNA damage, excess clonal hematopoiesis, and a distinct mutation profile in hematopoietic stem cells from aging Rassf5-/- mice compared with wildtype. We found sustained emergency granulopoiesis in Rassf5-/- mice, with repeated episodes accelerating AML, also similar to Irf8-/- mice. Identifying Nore1a downstream from Irf8 defines a pathway involved in leukemia suppression and the innate immune response and suggests a novel molecular mechanism contributing to age-related clonal myeloid disorders.

PRL2 phosphatase enhances oncogenic FLT3 signaling via dephosphorylation of the E3 ubiquitin ligase CBL at tyrosine 371.

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications-driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Dynamin-2 deficiency causes age- and sex-dependent neutropenia and myelodysplasia in mice.

The dynamins are a family of ubiquitously expressed GTPase proteins, best known for their role in membrane remodeling. Their contribution to hematopoiesis is incompletely recognized. Individuals with Charcot-Marie-Tooth disease with dynamin-2 (DNM2) mutations often develop neutropenia. We previously reported that dynamin (DNM) inhibition impairs SDF1a-mediated migration in megakaryocytes. Here, we report on conditionally Dnm2 deleted mice in hematopoietic tissues using the Vav-Cre murine strain. Homozygous Dnm2 deletion in blood tissues is embryonic lethal. Dnm2het male mice only developed a slightly decreased hemoglobin level. Dnm2het female mice developed leukopenia by 40 weeks of age and neutropenia by 65 weeks of age. Flow cytometry revealed decreased lineage-negative cells and granulocyte-monocyte progenitors in Dnm2het female mice. Immunohistochemical staining of bone marrow (BM) for mature neutrophils with Ly6G was decreased and myelodysplastic features were present in the BM of Dnm2het female mice. A linear distribution of Ly6G+ BM cells along blood vessels was observed in fewer Dnm2het mice than in controls, suggesting that the migration pattern in the marrow is altered. Marrow neutrophils treated with dynamin inhibitor, dynasore, showed increased cell surface CXCR4, suggesting that abnormal migration results in marrow neutrophil retention. Dnm2het female mice also developed splenomegaly secondary to germinal center hyperplasia at younger ages, suggesting perturbed immunity. In summary, female mice with BM Dnm2 haploinsufficiency developed neutropenia as they aged with decreased granulocyte progenitor production and migration defects. Our studies indicate a potential mechanism for the development of chronic idiopathic neutropenia, a disease that predominantly presents in middle-aged women.

Ruxolitinib ameliorates progressive anemia and improves survival during episodes of emergency granulopoiesis in Fanconi C-/- mice.

Fanconi anemia (FA) is an inherited disorder of DNA repair with hematologic manifestations that range from anemia to bone marrow failure to acute myeloid leukemia. In a murine model of FA (Fancc-/- mice), we found bone marrow failure was accelerated by repeated attempts to induce emergency (stress) granulopoiesis, the process for granulocyte production during the innate immune response. Fancc-/- mice exhibited an impaired granulocytosis response and died with profound anemia during repeated challenge. In the current study, we found erythropoiesis and serum erythropoietin decreased in Fancc-/- and wild-type (Wt) mice as emergency granulopoiesis peaked. Serum erythropoietin returned to baseline during steady-state resumption, and compensatory proliferation of erythroid progenitors was associated with DNA damage and apoptosis in Fancc-/- mice, but not Wt mice. The erythropoietin receptor activates Janus kinase 2 (Jak2), and we found treatment of Fancc-/- mice with ruxolitinib (Jak1/2-inhibitor) decreased anemia, enhanced granulocytosis, delayed clonal progression and prolonged survival during repeated emergency granulopoiesis episodes. This was associated with a decrease in DNA damage and apoptosis in Fancc-/- erythroid progenitors during this process. Transcriptome analysis of these cells identified enhanced activity of pathways for metabolism of reactive oxygen species, and decreased apoptosis- and autophagy-related pathways, as major ruxolitinib-effects in Fancc-/- mice. In contrast, ruxolitinib influenced primarily pathways involved in proliferation and differentiation in Wt mice. Ruxolitinib is approved for treatment of myeloproliferative disorders and graft-versus-host disease, suggesting the possibility of translational use as a bone marrow protectant in FA.

Inhibitory effects of Tomivosertib in acute myeloid leukemia.

The MAPK-interacting kinases 1 and 2 (MNK1/2) have generated increasing interest as therapeutic targets for acute myeloid leukemia (AML). We evaluated the therapeutic potential of the highly-selective MNK1/2 inhibitor Tomivosertib on AML cells. Tomivosertib was highly effective at blocking eIF4E phosphorylation on serine 209 in AML cells. Such inhibitory effects correlated with dose-dependent suppression of cellular viability and leukemic progenitor colony formation. Moreover, combination of Tomivosertib and Venetoclax resulted in synergistic anti-leukemic responses in AML cell lines. Mass spectrometry studies identified novel putative MNK1/2 interactors, while in parallel studies we demonstrated that MNK2 - RAPTOR - mTOR complexes are not disrupted by Tomivosertib. Overall, these findings demonstrate that Tomivosertib exhibits potent anti-leukemic properties on AML cells and support the development of clinical translational efforts involving the use of this drug, alone or in combination with other therapies for the treatment of AML.

An aberrantly sustained emergency granulopoiesis response accelerates postchemotherapy relapse in MLL1-rearranged acute myeloid leukemia in mice.

Acute myeloid leukemia (AML) with mixed lineage leukemia 1 (MLL1) gene rearrangement is characterized by increased expression of a set of homeodomain transcription factors, including homeobox A9 (HOXA9) and HOXA10. The target genes for these regulators include fibroblast growth factor 2 (FGF2) and Ariadne RBR E3 ubiquitin ligase 2 (ARIH2). FGF2 induces leukemia stem cell expansion in MLL1-rearranged AML. ARIH2 encodes TRIAD1, an E3 ubiquitin ligase required for termination of emergency granulopoiesis and leukemia suppressor function in MLL1-rearranged AML. Receptor tyrosine kinases (RTKs), including the FGF receptor, are TRIAD1 substrates that are possibly relevant to these activities. Using transcriptome analysis, we found increased activity of innate immune response pathways and RTK signaling in bone marrow progenitors from mice with MLL1-rearranged AML. We hypothesized that sustained RTK signaling, because of decreased TRIAD1 activity, impairs termination of emergency granulopoiesis during the innate immune response and contributes to leukemogenesis in this AML subtype. Consistent with this, we found aberrantly sustained emergency granulopoiesis in a murine model of MLL1-rearranged AML, associated with accelerated leukemogenesis. Treating these mice with an inhibitor of TRIAD1-substrate RTKs terminated emergency granulopoiesis, delayed leukemogenesis during emergency granulopoiesis, and normalized innate immune responses when combined with chemotherapy. Emergency granulopoiesis also hastened postchemotherapy relapse in mice with MLL1-rearranged AML, but remission was sustained by ongoing RTK inhibition. Our findings suggest that the physiological stress of infectious challenges may drive AML progression in molecularly defined subsets and identify RTK inhibition as a potential therapeutic approach to counteract this process.

Investigating the role of the innate immune response in relapse or blast crisis in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by expression of the tyrosine kinase oncogene, Bcr-abl. Tyrosine kinase inhibitors (TKI) induce prolonged remission in CML, and therapy discontinuation is an accepted approach to patients with reduction in Bcr-abl transcripts of four logs or greater. Half such individuals sustain a therapy free remission, but molecular mechanisms predicting relapse are undefined. We found relative calpain inhibition in CML cells with stabilization of calpain substrates, including ßcatenin and Xiap1. Since the Survivin gene is activated by ßcatenin, this identified two apoptosis-resistance mechanisms. We found that Survivin impaired apoptosis in leukemia stem cells (LSCs) and Xiap1 in CML granulocytes. Consistent with this, we determined treatment with an inhibitor of Survivin, but not Xiap1, prevented relapse during TKI treatment and after therapy discontinuation in a murine CML model. By transcriptome profiling, we identified activation of innate immune response pathways in murine CML bone marrow progenitors. This was increased by TKI treatment alone, but normalized with addition of a Survivin inhibitor. We found that activation of the innate immune response induced rapid blast crisis in untreated CML mice, and chronic phase relapse during a TKI discontinuation attempt. These results suggest that extrinsic stress exerts adverse effects on CML-LSCs.

Identification and targeting of novel CDK9 complexes in acute myeloid leukemia.

Aberrant activation of mTOR signaling in acute myeloid leukemia (AML) results in a survival advantage that promotes the malignant phenotype. To improve our understanding of factors that contribute to mammalian target of rapamycin (mTOR) signaling activation and identify novel therapeutic targets, we searched for unique interactors of mTOR complexes through proteomics analyses. We identify cyclin dependent kinase 9 (CDK9) as a novel binding partner of the mTOR complex scaffold protein, mLST8. Our studies demonstrate that CDK9 is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to RAPTOR and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to RICTOR, SIN1, and mLST8, forming CTORC2, and controls messenger RNA (mRNA) translation through phosphorylation of LARP1 and rpS6. Pharmacological targeting of CTORC complexes results in suppression of growth of primitive human AML progenitors in vitro and elicits strong antileukemic responses in AML xenografts in vivo.

Inhibitory effects of SEL201 in acute myeloid leukemia.

MAPK interacting kinase (MNK), a downstream effector of mitogen-activated protein kinase (MAPK) pathways, activates eukaryotic translation initiation factor 4E (eIF4E) and plays a key role in the mRNA translation of mitogenic and antiapoptotic genes in acute myeloid leukemia (AML) cells. We examined the antileukemic properties of a novel MNK inhibitor, SEL201. Our studies provide evidence that SEL201 suppresses eIF4E phosphorylation on Ser209 in AML cell lines and in primary patient-derived AML cells. Such effects lead to growth inhibitory effects and leukemic cell apoptosis, as well as suppression of leukemic progenitor colony formation. Combination of SEL201 with 5'-azacytidine or rapamycin results in synergistic inhibition of AML cell growth. Collectively, these results suggest that SEL201 has significant antileukemic activity and further underscore the relevance of the MNK pathway in leukemogenesis.

Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activity.

Megakaryocyte (MK) migration from the bone marrow periosteal niche toward the vascular niche is a prerequisite for proplatelet extension and release into the circulation. The mechanism for this highly coordinated process is poorly understood. Here we show that dynasore (DNSR), a small-molecule inhibitor of dynamins (DNMs), or short hairpin RNA knockdown of DNM2 and DNM3 impairs directional migration in a human MK cell line or MKs derived from cultured CD34+ cells. Because cell migration requires actin cytoskeletal rearrangements, we measured actin polymerization and the activity of cytoskeleton regulator RhoA and found them to be decreased after inhibition of DNM2 and DNM3. Because SDF-1α is important for hematopoiesis, we studied the expression of its receptor CXCR4 in DNSR-treated cells. CXCR4 expression on the cell surface was increased, at least partially because of slower endocytosis and internalization after SDF-1α treatment. Combined inhibition of DNM2 and DNM3 or forced expression of dominant-negative Dnm2-K44A or GTPase-defective DNM3 diminished ß1 integrin (ITGB1) activity. DNSR-treated MKs showed an abnormally clustered staining pattern of Rab11, a marker of recycling endosomes. This suggests decreased recruitment of the recycling pathway in DNSR-treated cells. Altogether, we show that the GTPase activity of DNMs, which governs endocytosis and regulates cell receptor trafficking, exerts control on MK migration toward SDF-1α gradients, such as those originating from the vascular niche. DNMs play a critical role in MKs by triggering membrane-cytoskeleton rearrangements downstream of CXCR4 and integrins.

Inhibition of Fas associated phosphatase 1 (Fap1) facilitates apoptosis of colon cancer stem cells and enhances the effects of oxaliplatin.

Fas associated phosphatase 1 (Fap1) is a ubiquitously expressed protein tyrosine phosphatase. Fap1 substrates include Fas and Gsk3ß, suggesting a role in regulating cell survival. Consistent with this, increased Fap1 expression is associated with resistance to Fas or platinum induced apoptosis in some human colon cancer tumors or cell lines. In the current studies, we found that Fap1 expression was significantly greater in CD133+ colon cancer stem cells compared to CD133- tumor cells. PTPN13 promoter activity (encoding Fap1) was repressed by interferon regulatory factor 2 (irf2), and expression of Fap1 and Irf2 were inversely correlated in CD133+ or CD133- colon cancer cells. We determined that CD133+ cells were relatively resistant to Fas or oxaliplatin induced apoptosis, but this was reversed by Fap1-knockdown or a Fap1-blocking tripeptide (SLV). In a murine xenograft model of colon cancer, we found treatment with SLV peptide significantly decreased tumor growth and relative abundance of CD133+CD44+ cells; associated with increased phosphorylation of Fap1 substrates. SLV peptide also enhanced inhibitory effects of oxaliplatin on tumor growth and Fap1 substrate phosphorylation in this model. Our studies suggest that therapeutically targeting Fap1 may decrease persistence of colon cancer stem cells during treatment with platinum chemotherapy by activating Fap1 substrates. In a murine model of chronic myeloid leukemia, we previously determined that inhibition of Fap1 decreased persistence of leukemia stem cells during tyrosine kinase inhibitor treatment. Therefore, Fap1 may be a tissue agnostic target to increase apoptosis in malignant stem cells.

The E3 ubiquitin ligase Triad1 influences development of Mll-Ell-induced acute myeloid leukemia.

Chromosomal translocations involving the MLL1 gene characterize a poor prognosis subset of acute myeloid leukemia (AML), referred to as 11q23-AML. Transcription of the HOXA9 and HOXA10 genes is enhanced in hematopoietic stem and progenitor cells in these leukemias. We previously found the ARIH2 gene was repressed by HoxA9 in myeloid progenitors, but activated by HoxA10 during granulopoiesis. ARIH2 encodes the Triad1 protein, an anti-proliferative E3 ubiquitin ligase. In the current study, we investigate the role of Triad1 in leukemogenesis induced by an MLL1 fusion protein (Mll-Ell). We found Mll-Ell increased expression of HoxA9, HoxA10, and Triad1 because HoxA9 represses only one of two ARIH2 cis elements that are activated by HoxA10. Although Triad1 antagonized the generally pro-proliferative effects of the Mll-Ell oncoprotein, we found blocking HoxA9 and HoxA10 phosphorylation shifted the balance to ARIH2 repression in Mll-Ell+ cells. We investigated the significance of these in vitro results in a murine bone marrow transplant model. We found Triad1 knockdown significantly shortened the latency to development of AML in mice transplanted with Mll-Ell-transduced bone marrow. And, Triad1 expression fell during the prolonged AML latency period in mice transplanted with bone marrow expressing Mll-Ell alone. Our studies identify Triad1 as a leukemia suppressor in 11q23-AML. This suggests defining relevant Triad1 substrates may indicate novel therapeutic targets in this disease.

TP53 Haploinsufficiency Rescues Emergency Granulopoiesis in FANCC-/- Mice.

Emergency (stress) granulopoiesis is an episodic process for the production of granulocytes in response to infectious challenge. We previously determined that Fanconi C, a component of the Fanconi DNA-repair pathway, is necessary for successful emergency granulopoiesis. Fanconi anemia results from mutation of any gene in this pathway and is characterized by bone marrow failure (BMF) in childhood and clonal progression in adolescence. Although murine Fanconi anemia models exhibit relatively normal steady-state hematopoiesis, FANCC-/- mice are unable to mount an emergency granulopoiesis response. Instead, these mice develop BMF and die during repeated unsuccessful emergency granulopoiesis attempts. In FANCC-/- mice, BMF is associated with extensive apoptosis of hematopoietic stem and progenitor cells through an undefined mechanism. In this study, we find that TP53 haploinsufficiency completely rescues emergency granulopoiesis in FANCC-/- mice and protects them from BMF during repeated emergency granulopoiesis episodes. Instead, such recurrent challenges accelerated clonal progression in FANCC-/-TP53+/- mice. In FANCC-/- mice, BMF during multiple emergency granulopoiesis attempts was associated with increased ataxia telangiectasia and Rad3-related protein (Atr) and p53 activation with each attempt. In contrast, we found progressive attenuation of expression and activity of Atr, and consequent p53 activation and apoptosis, in the bone marrow of FANCC-/-TP53+/- mice during this process. Therefore, activation of Atr-with consequent Fanconi-mediated DNA repair or p53-dependent apoptosis-is an essential component of emergency granulopoiesis and it protects the bone marrow from genotoxic stress during this process.

Stat3 and CCAAT enhancer-binding protein ß (C/ebpß) activate Fanconi C gene transcription during emergency granulopoiesis.

Interferon consensus sequence-binding protein (Icsbp) is required for terminating emergency granulopoiesis, an episodic event responsible for granulocyte production in response to infections and a key component of the innate immune response. Icsbp inhibits the expression of Stat3 and C/ebpß, transcription factors essential for initiating and sustaining granulopoiesis, and activates transcription of Fanconi C (FANCC), a DNA repair protein. In prior studies, we noted accelerated bone marrow failure in Fancc-/- mice undergoing multiple episodes of emergency granulopoiesis, associated with apoptosis of bone marrow cells with unrepaired DNA damage. Additionally, we found increased expression of Fanconi C and F proteins during emergency granulopoiesis. These findings suggest that Icsbp protects the bone marrow from DNA damage by increasing activity of the Fanconi DNA repair pathway, but the mechanisms for FANCC activation during initiation of emergency granulopoiesis are unclear. In this study, we observed that Stat3 and C/ebpß activate FANCC transcription and contribute to DNA repair. Our findings indicate that FancC expression is increased during Stat3- and C/ebpß-induced initiation of emergency granulopoiesis by these transcription factors and is maintained through termination by Icsbp. Our work reveals that Stat3- and C/ebpß-mediated FancC expression is a critical component for initiating and sustaining key innate immune responses.

Decreased calpain activity in chronic myeloid leukemia impairs apoptosis by increasing survivin in myeloid progenitors and xiap1 in differentiating granulocytes.

Chronic Myeloid Leukemia (CML) is characterized by translocations between chromosomes 9 and 22, resulting in expression of Bcr-abl oncogenes. Although the clinical course of CML was revolutionized by development of Bcr-abl-directed tyrosine kinase inhibitors (TKIs), CML is not cured by these agents. Specifically, the majority of subjects relapsed in clinical trials attempting TKI discontinuation, suggesting persistence of leukemia stem cells (LSCs) even in molecular remission. Identifying mechanisms of CML-LSC persistence may suggest rationale therapeutic targets to augment TKI efficacy and lead to cure. Apoptosis resistance is one proposed mechanism. In prior studies, we identified increased expression of Growth Arrest Specific 2 (Gas2; a Calpain inhibitor) in Bcr-abl+ bone marrow progenitor cells. A number of previously described Calpain substrates might influence apoptosis in CML, including ßcatenin and the X-linked Inhibitor of Apoptosis Protein 1 (Xiap1). We previously found Gas2/Calpain dependent stabilization of ßcatenin in CML, and increased expression of ßcatenin target genes, including Survivin (also an IAP). In the current work, we investigate contributions of Survivin and Xiap1 to Fas-resistance in Bcr-abl+ bone marrow cells. Inhibitors of these proteins are currently in clinical trials for other malignancies, but a role for either IAP in CML-LSC persistence is unknown.

Central Regulatory Role for SIN1 in Interferon γ (IFNγ) Signaling and Generation of Biological Responses.

The precise signaling mechanisms by which type II IFN receptors control expression of unique genes to induce biological responses remain to be established. We provide evidence that Sin1, a known element of the mammalian target of rapamycin complex 2 (mTORC2), is required for IFNγ-induced phosphorylation and activation of AKT and that such activation mediates downstream regulation of mTORC1 and its effectors. These events play important roles in the assembly of the eukaryotic translation initiation factor 4F (eIF4F) and mRNA translation of IFN-stimulated genes. Interestingly, IFNγ-induced tyrosine phosphorylation of STAT1 is reduced in cells with targeted disruption of Sin1, leading to decreased transcription of several IFNγ-inducible genes in an mTORC2-independent manner. Additionally, our studies establish that Sin1 is essential for generation of type II IFN-dependent antiviral effects and antiproliferative responses in normal and malignant hematopoiesis. Together, our findings establish an important role for Sin1 in both transcription and translation of IFN-stimulated genes and type II IFN-mediated biological responses, involving both mTORC2-dependent and -independent functions.

Bcr-abl regulates Stat5 through Shp2, the interferon consensus sequence binding protein (Icsbp/Irf8), growth arrest specific 2 (Gas2) and calpain.

Icsbp/Irf8 is an interferon regulatory transcription factor that functions as a suppressor of myeloid leukemias. Consistent with this activity, Icsbp represses a set of genes encoding proteins that promote cell proliferation/survival. One such gene encodes Gas2, a calpain inhibitor. We previously found that increased Gas2-expression in Bcr-abl+ cells stabilized ßcatenin; a Calpain substrate. This was of interest, because ßcatenin contributes to disease progression in chronic myeloid leukemia (CML). Calpain has additional substrates implicated in leukemogenesis, including Stat5. In the current study, we hypothesized that Stat5 activity in CML is regulated by Gas2/Calpain. We found that Bcr-abl-induced, Shp2-dependent dephosphorylation of Icsbp impaired repression of GAS2 by this transcription factor. The consequent decrease in Calpain activity stabilized Stat5 protein; increasing the absolute abundance of both phospho and total Stat5. This enhanced repression of the IRF8 promoter by Stat5 in a manner dependent on Icsbp, Gas2 and Calpain, but not Stat5 tyrosine phosphorylation. During normal myelopoiesis, increased expression and phosphorylation of Icsbp inhibits Calpain. In contrast, constitutive activation of Shp2 in Bcr-abl+ cells impairs regulation of Gas2/Calpain by Icsbp, aberrantly stabilizing Stat5 and enhancing IRF8 repression. This novel feedback mechanism enhances leukemogenesis by increasing Stat5 and decreasing Icsbp. Bcr-abl targeted tyrosine kinase inhibitors (TKIs) provide long term disease control, but CML is not cured by these agents. Our studies suggest targeting Calpain might be a rational therapeutic approach to decrease persistent leukemia stem cells (LSCs) during TKI-treatment.

Cooperation between AlphavBeta3 integrin and the fibroblast growth factor receptor enhances proliferation of Hox-overexpressing acute myeloid leukemia cells.

A poor prognosis subtype of acute myeloid leukemia (AML) is characterized by increased expression of a set of homeodomain (HD) transcription factors, including HoxA9, HoxA10 and Cdx4. This encompasses AML with MLL1 gene translocations, because Mll1-fusion proteins aberrantly activate HOX transcription. We previously identified FGF2 (Fibroblast Growth Factor 2) as a target gene for HoxA9 and HoxA10 that was indirectly activated by Mll-Ell (an Mll1-fusion protein). Autocrine stimulation of Mll-Ell+ myeloid progenitor cells by Fgf2 stabilized ßcatenin and increased expression of ßcatenin target genes, including CDX4. Since HOXA9 and HOXA10 are Cdx4 target genes, Fgf2 indirectly augmented direct effects of Mll-Ell on these genes. ITGB3, encoding ß3 integrin, is another HoxA10 target gene. In the current studies, we found activation of ITGB3 transcription in Mll-Ell+ myeloid progenitor cells via HoxA9 and HoxA10. Increased expression of αvß3 integrin increased Syk-activation; contributing to cytokine hypersensitivity. However, inhibiting Fgf-R partly reversed αvß3 activity in Mll-Ell+ progenitor cells by decreasing ITGB3 promoter activity in a ßcatenin- and Cdx4-dependent manner. Inhibitors of Fgf-R or Syk impaired proliferation of CD34+ bone marrow cells from AML subjects with increased Hox-expression; with a greater combined effect. These studies identified a rational therapeutic approach to this AML subtype.