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Supplementary Figures and Legends

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Supplementary Table 4

Supplementary Table 1

Core-shell ceramics fibers with multiple composition are ideal candidates for thermal insulation application at high temperature. Herein, ultrathin C–SiC/ZrSixOyCz core-shell fibers were successfully fabricated by coaxial electrospinning and high temperature pyrolysis with polycarbosilane (PCS) as core precursor and Zirconium butoxide as shell precursor, respectively. The ceramics fibers revealed distinct core-shell partition. The average diameter was about 3 μm and the shell thickness increased to 500 nm with increasing pyrolysis temperature. Thermogravimetry analysis (TGA) results showed that the weight of the fibers reduced to 60% in Ar atmosphere and rose to 108% in O2 atmosphere, which confirmed the excellent thermal stability and anti-oxidation properties due to the protection effect of shell layer and the self-healing mechanism. Moreover, the ceramics fibers maintained good flexibility and deformability. Such optimized anti-oxidation and mechanical performance indicates that the prepared core-shell fibers could be an excellent thermal insulation material for high temperature application.

Introduction: Iberdomide (IBER; CC-220) is a potent, novel cereblon (CRBN) E3 ligase modulator (CELMoD) agent under investigation in a phase 1/2 dose-escalation study in relapsed/refractory multiple myeloma (MM) (CC-220-MM-001; NCT02773030). IBER modulates CRBN to induce ubiquitination and proteasome-dependent degradation of Ikaros/Aiolos, resulting in anti-myeloma and immunomodulatory activity. Compared with immunomodulatory drugs (IMiDs), IBER binds to CRBN with 20-fold higher affinity and is more efficient at degrading Ikaros/Aiolos. Additionally, IBER can overcome IMiD drug resistance. Here, we evaluate the preclinical activity of IBER in combination with monoclonal antibodies (mAbs) and assess immune pharmacodynamic changes of IBER + daratumumab (DARA) among patients in the CC-220-MM-001 study. Methods: Preclinical analyses were performed on MM cell lines and peripheral blood mononuclear cells (PBMCs) from healthy volunteers. In co-culture assays, cell lines and immune cells were treated with clinically relevant concentrations of lenalidomide (LEN; 1 μM), pomalidomide (POM; 300 nM), and IBER (20 nM) alone, and in combination with DARA or elotuzumab (ELO). Clinical biomarker data were obtained from blood samples of patients enrolled in IBER + dexamethasone (DEX) and IBER + DARA + DEX cohorts of the CC-220-MM-001 study. Immune profiling was evaluated by flow cytometry on Cycle (C) 1 Day (D) 1, C2D15, C4D1, and C4D15. Results: Treatment of CD3-stimulated PBMCs with IBER increased cytokine secretion more potently than with IMiD drugs, promoting natural killer (NK) cell proliferation and increasing NK cell numbers by > 30% in cultures. Treatment of CD3-stimulated PBMCs with IBER also promoted immune-mediated killing of MM cell lines, and pretreatment of MM cell lines for 48 h followed by washout sensitized cells to immune-mediated clearance. IBER treatment of MM cells increased protein expression of CD38, but had no discernable effect on SLAMF7 expression. IBER in combination with DARA or ELO enhanced the immune-mediated killing of MM cell lines and resulted in deeper cell killing compared with LEN or POM when combined with the same mAbs. Analogous to preclinical data, immune profiling of patients treated with IBER + DEX demonstrated an immune-stimulatory effect, including increased proliferating NK and T cells. Notably, patients coming on trial directly after a DARA-containing regimen showed depleted NK cell counts, which rebounded by nearly 2-fold after 1 cycle of IBER + DEX. Additionally, the effects of IBER on T and NK cell proliferation were similar in patients treated with IBER + DEX + DARA and IBER + DEX, except for overall reduction in CD38-expressing NK and T cells in patients receiving DARA. Conclusions: IBER induced immune-stimulatory activity more potently than LEN or POM, enhancing cytokine release and NK cell proliferation. MM cells treated with IBER were also sensitized to immune-mediated clearance. These data suggest that IBER may enhance the activity of biologics that exert anti-malignant activity through antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Accordingly, IBER enhanced the activity of DARA and ELO in co-culture. IBER also induced an immune-stimulatory effect on NK and T cells in vivo, confirming immune-stimulatory effects of IBER in patients with MM. Notably, the effect of IBER on NK cells was more pronounced in patients whose last treatment regimen contained DARA, suggesting IBER treatment may contribute to NK cell recovery after DARA treatment. Furthermore, immune changes observed by the addition of DARA to IBER + DEX were similar, suggesting IBER was the primary agent promoting immunomodulation in this triplet therapy. Together, these data support continued clinical development of IBER in combination with CD38- and SLAMF7-directed mAbs as well as other immune-directed therapies for the treatment of MM. Disclosures Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ma:Bristol Myers Squibb: Current Employment. Wollerman:Bristol Myers Squibb: Current Employment. Pierceall:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lonial:JUNO Therapeutics: Consultancy; Merck: Consultancy, Honoraria, Other: Personal fees; Abbvie: Consultancy; GSK: Consultancy, Honoraria, Other: Personal fees; BMS: Consultancy, Honoraria, Other: Personal fees, Research Funding; Novartis: Consultancy, Honoraria, Other: Personal fees; Takeda: Consultancy, Other: Personal fees, Research Funding; Genentech: Consultancy; Karyopharm: Consultancy; Sanofi: Consultancy; Amgen: Consultancy, Honoraria, Other: Personal fees; Janssen: Consultancy, Honoraria, Other: Personal fees, Research Funding; Onyx: Honoraria; Millennium: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees. van de Donk:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ferrer: Membership on an entity's Board of Directors or advisory committees. Popat:Celgene: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding; AbbVie: Consultancy, Honoraria; GSK: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Jagannath:BMS, Janssen, Karyopharm, Legend Biotech, Sanofi, Takeda: Consultancy. Zonder:Intellia: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; BMS: Consultancy, Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Celgene: Research Funding; Prothena: Consultancy; Janssen: Consultancy, Other: Personal fees; Oncopeptide: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Caelum: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees. Minnema:Kite, a Gilead Company: Speakers Bureau; Servier: Consultancy; Amgen: Consultancy; Celgene: Other: travel support, Research Funding. Oriol:Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Larsen:Janssen Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Badros:Amgen: Consultancy; University of Maryland: Current Employment. Rodriguez-Otero:Sanofi: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); GlaxoSmithKline: Consultancy, Current Employment, Current equity holder in publicly-traded company, Honoraria; Medscape: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Kite: Consultancy, Honoraria. Sonneveld:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Skyline Dx: Honoraria, Research Funding; Sanofi: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Nguyen:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hong:Bristol Myers Squibb: Current Employment. Sorrell:Bristol Myers Squibb: Current Employment; Children's Oncology Group: Other: Non-member; Previous Study Chair AAML08B1. Thakurta:Bristol Myers Squibb: Current Employment; Oxford University: Other: Visiting professor.

C/EBPα plays a key role in specifying myeloid lineage development. HoxA9 is expressed in myeloid progenitors, with its level diminishing during myeloid maturation, and HOXA9 is over-expressed in a majority of acute myeloid leukemia cases, including those expressing NUP98-HOXD13. The objective of this study was to determine whether HoxA9 directly represses Cebpa gene expression. We find 4-fold increased HoxA9 and 5-fold reduced Cebpa in marrow common myeloid and LSK progenitors from Vav-NUP98-HOXD13 transgenic mice. Conversely, HoxA9 decreases 5-fold while Cebpa increases during granulocytic differentiation of 32Dcl3 myeloid cells. Activation of exogenous HoxA9-ER in 32Dcl3 cells reduces Cebpa mRNA even in the presence of cycloheximide, suggesting direct repression. Cebpa transcription in murine myeloid cells is regulated by a hematopoietic-specific +37 kb enhancer and by a more widely active +8 kb enhancer. ChIP-Seq analysis of primary myeloid progenitor cells expressing exogenous HoxA9 or HoxA9-ER demonstrates that HoxA9 localizes to both the +8 kb and +37 kb Cebpa enhancers. Gel shift analysis demonstrates HoxA9 binding to three consensus sites in the +8 kb enhancer, but no affinity for the single near-consensus site present in the +37 kb enhancer. Activity of a Cebpa +8 kb enhancer/promoter-luciferase reporter in 32Dcl3 or MOLM14 myeloid cells is increased ~2-fold by mutation of its three HOXA9-binding sites, suggesting that endogenous HoxA9 represses +8 kb Cebpa enhancer activity. In contrast, mutation of five C/EBPα-binding sites in the +8 kb enhancer reduces activity 3-fold. Finally, expression of a +37 kb enhancer/promoter-hCD4 transgene reporter is reduced ~2-fold in marrow common myeloid progenitors when the Vav-NUP98-HOXD13 transgene is introduced. Overall, these data support the conclusion that HoxA9 represses Cebpa expression, at least in part via inhibition of its +8 kb enhancer, potentially allowing normal myeloid progenitors to maintain immaturity and contributing to the pathogenesis of acute myeloid leukemia associated with increased HOXA9.

Despite the important role of the PI3K/AKT/mTOR axis in the pathogenesis of cancer, to date there have been few functional oncogenic fusions identified involving the AKT genes. A 12-year-old female with a histopathologically indeterminate epithelioid neoplasm was found to harbor a novel fusion between the LAMTOR1 and AKT1 genes. Through expanded use access, she became the first pediatric patient to be treated with the oral ATP-competitive pan-AKT inhibitor ipatasertib. Treatment resulted in dramatic tumor regression, demonstrating through patient-driven discovery that the fusion resulted in activation of AKT1, was an oncogenic driver, and could be therapeutically targeted with clinical benefit. Post-clinical validation using patient-derived model systems corroborated these findings, confirmed a membrane-bound and constitutively active fusion protein, and identified potential mechanisms of resistance to single-agent treatment with ipatasertib. Significance: This study describes the patient-driven discovery of the first AKT1 fusion–driven cancer and its treatment with the AKT inhibitor ipatasertib. Patient-derived in vitro and in vivo model systems are used to confirm the LAMTOR1–AKT1 fusion as a tumorigenic driver and identify potential mechanisms of resistance to AKT inhibition. This article is highlighted in the In This Issue feature, p. 565

Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E–binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9–positive leukemic stem cells by modulating AKT and 4E-BP1 phosphorylation. Taken together, these results suggest that S6K1 acts through multiple targets of the mTOR pathway to promote self-renewal and leukemia progression. Given the recent interest in S6K1 as a potential therapeutic target in cancer, our results further support targeting this molecule as a potential strategy for treatment of myeloid malignancies.

PRDM16 is a transcription co-factor that plays critical roles in development of brown adipose tissue, as well as maintenance of adult hematopoietic and neural stem cells. Here we report that PRDM16 is a histone H3K4 methyltransferase on chromatin. Mutation in the N-terminal PR domain of PRDM16 abolishes the intrinsic enzymatic activity of PRDM16. We show that the methyltransferase activity of PRDM16 is required for specific suppression of MLL fusion protein-induced leukemogenesis both in vitro and in vivo. Mechanistic studies show that PRDM16 directly activates the SNAG family transcription factor Gfi1b, which in turn downregulates the HOXA gene cluster. Knockdown Gfi1b represses PRDM16-mediated tumor suppression, while Gfi1b overexpression mimics PRDM16 overexpression. In further support of the tumor suppressor function of PRDM16, silencing PRDM16 by DNA methylation is concomitant with MLL-AF9-induced leukemic transformation. Taken together, our study reveals a previously uncharacterized function of PRDM16 that depends on its PR domain activity.

The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis., The interaction of DOT1L with MLL oncogenic fusion proteins has been implicated in leukemogenesis. Here, the authors show a contrasting role for DOT1L in protecting UVR-induced melanomagenesis by facilitating DNA repair through interaction with XPC.

While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.

Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.

Key points • We investigated how the F1486 deletion LQT3 mutation impairs the functional properties of the human cardiac voltage-gated sodium channel (hNav1.5) and alters action potential firing. • Voltage-clamp recordings from HEK 293 cells and cardiomyocytes expressing recombinant channels demonstrated that the F1486del mutation reduces peak current density but also impairs inactivation and increases late current density. • Current-clamp recordings from cardiomyocytes indicated that the increase in late current density would result in prolonged action potential duration and this was confirmed using computer simulations. • The deletion of F1486 abolished the ability of lidocaine to stabilize the inactivated state and eliminated the high-affinity binding of lidocaine to inactivated channels. • Our data show that the hNav1.5-F1486del mutation has complex functional consequences and indicate that knowledge of the specific molecular defect is critical when developing potential treatments for individuals with prolonged QT intervals. Abstract The deletion of phenylalanine 1486 (F1486del) in the human cardiac voltage-gated sodium channel (hNav1.5) is associated with fatal long QT (LQT) syndrome. In this study we determined how F1486del impairs the functional properties of hNav1.5 and alters action potential firing in heterologous expression systems (human embryonic kidney (HEK) 293 cells) and their native cardiomyocyte background. Cells expressing hNav1.5-F1486del exhibited a loss-of-function alteration, reflected by an 80% reduction of peak current density, and several gain-of-function alterations, including reduced channel inactivation, enlarged window current, substantial augmentation of persistent late sodium current and an increase in ramp current. We also observed substantial action potential duration (APD) prolongation and prominent early afterdepolarizations (EADs) in neonatal cardiomyocytes expressing the F1486del channels, as well as in computer simulations of myocyte activity. In addition, lidocaine sensitivity was dramatically reduced, which probably contributed to the poor therapeutic outcome observed in the patient carrying the hNav1.5-F1486del mutation. Therefore, despite the significant reduction in peak current density, the F1486del mutation also leads to substantial gain-of-function alterations that are sufficient to cause APD prolongation and EADs, the predominant characteristic of LQTs. These data demonstrate that hNav1.5 mutations can have complex functional consequences and highlight the importance of identifying the specific molecular defect when evaluating potential treatments for individuals with prolonged QT intervals.

The growth and maturation of bone marrow-derived mast cells (BMMCs) from precursors are regulated by coordinated signals from multiple cytokine receptors, including KIT. While studies conducted using mutant forms of these receptors lacking the binding sites for Src family kinases (SFKs) and phosphatidylinositol-3-kinase (PI3K) suggest a role for these signaling molecules in regulating growth and survival, how complete loss of these molecules in early BMMC progenitors (MCps) impacts maturation and growth during all phases of mast cell development is not fully understood. We show that the Lyn SFK and the p85α subunit of class I(A) PI3K play opposing roles in regulating the growth and maturation of BMMCs in part by regulating the level of PI3K. Loss of Lyn in BMMCs results in elevated PI3K activity and hyperactivation of AKT, which accelerates the rate of BMMC maturation due in part to impaired binding and phosphorylation of SHIP via Lyn's unique domain. In the absence of Lyn's unique domain, BMMCs behave in a manner similar to that of Lyn- or SHIP-deficient BMMCs. Importantly, loss of p85α in Lyn-deficient BMMCs not only represses the hyperproliferation associated with the loss of Lyn but also represses their accelerated maturation. The accelerated maturation of BMMCs due to loss of Lyn is associated with increased expression of microphthalmia-associated transcription factor (Mitf), which is repressed in MCps deficient in the expression of both Lyn and p85α relative to controls. Our results demonstrate a crucial interplay of Lyn, SHIP, and p85α in regulating the normal growth and maturation of BMMCs, in part by regulating the activation of AKT and the expression of Mitf.

Mast cell maturation is poorly understood. We show that enhanced PI3K activation results in accelerated maturation of mast cells by inducing the expression of microphthalmia transcription factor (Mitf). Conversely, loss of PI3K activation reduces the maturation of mast cells by inhibiting the activation of AKT, leading to reduced Mitf but enhanced Gata-2 expression and accumulation of Gr1+Mac1+ myeloid cells as opposed to mast cells. Consistently, overexpression of Mitf accelerates the maturation of mast cells, whereas Gata-2 overexpression mimics the loss of the PI3K phenotype. Expressing the full-length or the src homology 3– or BCR homology domain–deleted or shorter splice variant of the p85α regulatory subunit of PI3K or activated AKT or Mitf in p85α-deficient cells restores the maturation but not growth. Although deficiency of both SHIP and p85α rescues the maturation of SHIP−/− and p85α−/− mast cells and expression of Mitf; in vivo, mast cells are rescued in some, but not all tissues, due in part to defective KIT signaling, which is dependent on an intact src homology 3 and BCR homology domain of p85α. Thus, p85α-induced maturation, and growth and survival signals, in mast cells can be uncoupled.

Objective Src family kinases (SFK) have been implicated in regulating growth factor and integrin-induced proliferation, migration, and gene expression in multiple cell types. However, little is known about the role of these kinases in the growth, homing, and engraftment potential of hematopoietic stem and progenitor cells. Results Here we show that loss of hematopoietic-specific SFKs Hck, Fgr, and Lyn results in increased number of Sca-1 + Lin − cells in the bone marrow, which respond differentially to cytokine-induced growth in vitro and manifest a significant defect in the long-term repopulating potential in vivo. Interestingly, a significant increase in expression of adhesion molecules, known to coincide with the homing potential of wild-type bone marrow cells is also observed on the surface of SFK −/− cells, although, this increase did not affect the homing potential of more primitive Lin − Sca-1 + SFK −/− cells. The stem cell−repopulating defect observed in mice transplanted with SFK −/− bone marrow cells is due to the loss of Lyn Src kinase, because deficiency of Lyn, but not Hck or Fgr, recapitulated the long-term stem cell defect observed in mice transplanted with SFK −/− bone marrow cells. Conclusions Taken together, our results demonstrate an essential role for Lyn kinase in positively regulating the long-term and multilineage engraftment of stem cells, which is distinct from its role in mature B cells and myeloid cells.

Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that is essential for hematopoietic stem cell expansion and differentiation. Deregulation of HOXA9 is commonly observed in human acute myeloid leukemia (AML). About half of AML patients overexpress HOXA9 as a result of MLL rearrangements, NUP98 translocations, NPM1 mutations or CDX2/CDX4 overexpression. Despite its central importance in leukemia, the mechanism of transcriptional regulation by HOXA9 and its downstream effectors are poorly understood. HOXA9 physically interacts with MEIS1, a cofactor that greatly accelerates leukemia development in transplanted animals. Our group recently identified a number of transcription factors as HOXA9 potential collaborators by genomic profiling of HOXA9 binding sites and mass spectroscopy. One of these putative collaborators is signal transducer and activator of transcription 5 (STAT5), which coimmunoprecipitates with HOXA9. Furthermore STAT motifs extensively overlap with HOXA9 binding sites. STAT5 is important for survival, proliferation and differentiation of hematopoietic cells and constitutive activation of STAT5 has also been observed in human leukemias bearing oncogenic mutation of Jak2, Bcr-Abl, c-Kit and Flt3. FLT3 internal tandem duplication (FLT3-ITD) is observed in 25% of patients with MLL-partial tandem duplication (MLL-PTD) and is associated with HOXA9 upregulation and unfavorable prognosis. Therefore, we hypothesized that the interaction of HOXA9 and STAT5 may play a role in HOXA9-associated leukemogenesis. Treatment of human cell lines bearing MLL-AF9 and FLT3-ITD with specific FLT3 and STAT5 inhibitors showed that suppression of the constitutive activation of STAT5 significantly inhibits the hyper-proliferation of these cells. We then overexpressed FLT3-ITD or active mutation of STAT5 (STAT5 1*6) in mouse hematopoietic stem cells /progenitor cells (HSC/PCs) transduced with MLL-AF9 or HOXA9 and found that constitutively active STAT5 enhances cell proliferation in vitro. We next transduced HOXA9 into HSC/Pcs from wild type (WT) or FLT3-ITD transgenic mice and transplanted these cells into sublethally irradiated WT mice. All of these recipients developed myeloid leukemia, with recipients transplanted with FLT3-ITD (n=4) developing leukemia significantly earlier than WT controls (n=5, p To further assess the role of STAT5 in HOXA9-mediated transformation, we performed ChIP-Seq assay with HOXA9-transformed cells and identified nearly half of STAT5 binding sites (228 out of 596) colocalized with HOXA9. Most of these cobound sites are located in distal intergenic (61.0%) and intron (35.1%) regions. Five cobound regions (Il2rα, Fgf1, Pdlim5, Pim1, Fabp5) were selected and confirmed by ChIP-qPCR. To further characterize the interaction between HOXA9 and STAT5, GST pull-down assays were performed that showed that the c-terminal of HOXA9 is critical for interaction with STAT5. Overall, the findings suggest that STAT5 promotes HOXA9-induced transformation by functionally interacting with HOXA9 at HOXA9-regulated enhancers. Disclosures No relevant conflicts of interest to declare.

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21–activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.

Erythropoiesis is a dynamic, multistep process whereby hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Although several downstream signaling molecules have been identified that regulate steady-state erythropoiesis, the major regulators under conditions of stress remain poorly defined. Rho kinases (ROCKs) belong to a family of serine/threonine kinases. Using gene-targeted ROCK1-deficient mice, we show that lack of ROCK1 in phenylhydrazine-induced oxidative stress model results in enhanced recovery from hemolytic anemia as well as enhanced splenic stress erythropoiesis compared with control mice. Deficiency of ROCK1 also results in enhanced survival, whereas wild-type mice die rapidly in response to stress. Enhanced survivability of ROCK1-deficient mice is associated with reduced level of reactive oxygen species. BM transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous. We show that ROCK1 binds to p53 and regulates its stability and expression. In the absence of ROCK1, p53 phosphorylation and expression is significantly reduced. Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia.

We show that loss of p85α inhibits the growth and maturation of mast cells, whereas loss of p85β enhances this process. Whereas restoring the expression of p85α in P85α−/− cells restores these functions, overexpression of p85β has the opposite effect. Consistently, overexpression of p85β in WT mast cells represses KIT-induced proliferation and IL-3–mediated maturation by inhibiting the expression of Microphthalmia transcription factor. Because p85α and p85β differ in their N-terminal sequences, chimeric proteins consisting of amino or carboxy-terminal of p85α and/or p85β do not rescue the growth defects of p85α−/− cells, suggesting cooperation between these domains for normal mast cell function. Loss of p85β impaired ligand induced KIT receptor internalization and its overexpression enhanced this process, partly because of increased binding of c-Cbl to p85β relative to p85α. In vivo, loss of p85β resulted in increased mast cells, and bone marrow transplantation of cells overexpressing p85β resulted in significant reduction in some tissue mast cells. Overexpression of p85β suppressed the growth of oncogenic KIT-expressing cells in vitro and prolonged the survival of leukemic mice in vivo. Thus, p85α and p85β differentially regulate SCF and oncogenic KIT-induced signals in myeloid lineage-derived mast cells.

Stem cell factor (SCF) mediated KIT receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from KIT are poorly understood. Mast cells express multiple regulatory subunits of class 1(A) PI3Kinase (PI3K) including p85α, p85β, p50α, and p55α. While it is known that PI3K plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including growth, survival and cycling are not known. We show that loss of p85α impairs the growth, survival and cycling of mast cell progenitors (MCp). To delineate the molecular mechanism (s) by which p85α regulates mast cell growth, survival and cycling, we performed microarray analyses to compare the gene expression profile of MCps derived from WT and p85α-deficient mice in response to SCF stimulation. We identified 151 unique genes exhibiting altered expression in p85α-deficient cells in response to SCF stimulation compared to WT cells. Functional categorization based on DAVID bioinformatics tool and Ingenuity Pathway Analysis (IPA) software relates the altered genes due to lack of p85α to transcription, cell cycle, cell survival, cell adhesion, cell differentiation, and signal transduction. Our results suggest that p85α is involved in mast cell development through regulation of expression of growth, survival and cell cycle related genes.

The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult hematopoietic stem cells (HSCs) and regulation of progenitor population. Translocations in MLL have been detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. Various genes, including AF4, AF5, AF9, ELL and ENL have been identified as partners for translocation in MLL-rearranged leukemia. In hematopoietic cells, expression of MLL fusion proteins result in a block of differentiation. AML patients who successfully undergo treatment but relapse suggest the importance of targeting leukemia initiating cells (LICs) within the MLL leukemia. LICs remain in a quiescent state and are capable of survival despite treatment with chemotherapeutic agents or targeted molecular inhibitors. In mouse models, LICs are defined by the capability of successfully propagating disease upon serial transplantation. In order to prevent disease relapse, identification of molecules, which regulate the self-renewal of LICs, is essential. The phosphatidylinositol 3-kinase (PI3K)-Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway is a key regulator of self-renewal of both HSCs and LICs. Deletion of Raptor, a subunit of mTORC1, does not affect initiation and progression of acute myeloid leukemia (AML), but Raptor deficiency results in delayed propagation of AML. Upon its activation, mTORC1 phosphorylates and activates p70 ribosomal protein S6 kinase (S6K1) and inhibits the activity of eukaryote translation initiation factor 4E binding protein 1 (4E-BP1). S6K1 has been shown to be hyperactivated in hematopoietic cells expressing oncogenic MLL-AF9 fusion protein. In our study, we have assessed the role of S6K1 in the initiation, progression and propagation of AML using a genetic model of S6K1 knockout mice (S6K1-/-). We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and transplanted them into lethally irradiated recipients. Recipients of both WT and S6K1-/- HSC/Ps bearing MLL-AF9 displayed high white blood cell (WBC) count, splenomegaly and developed AML. There was no difference in survival between the WT and S6K1-/- recipients. In order to determine whether S6K1 regulates the self-renewal of LICs, we transplanted lethally irradiated mice with cells from WT and S6K1-/- primary recipients who developed AML. Recipients of S6K1 deficient AML cells survived significantly longer compared to controls (n=17/group, p To determine the role of S6K1 in normal HSC development and maintenance, we analyzed bone marrow derived HSCs in WT and S6K1-/- mice. S6K1 deficiency did not alter the frequency of long term HSCs (LT-HSCs) as defined by CD150+ CD48- Lin- Sca1+ c-Kit+ surface markers, but the absolute number of LT-HSCs were significantly reduced in S6K1 deficient mice (p Disclosures No relevant conflicts of interest to declare.

Regulation of hematopoietic stem cell (HSC) function(s) via the mammalian target of rapamycin complex1 (mTORC1) and its upstream regulators including PI3K and Akt has been described before. To this end, we and others have shown that hyperactivation and deficiency of the PI3K-mTORC1 pathway results in altered development, maintenance and function(s) of HSCs. However, the role of downstream effector of mTORC1, p70S6 kinase (S6K1), in HSC development and functions is unknown. Previous studies have implicated S6K1 as a regulator of ageing, by virtue of its ability to regulate cellular metabolic processes as well as protein translation. In certain cells, however S6K1 regulates cell survival and also acts as a negative regulator of PI3K-mTORC1 pathway, thus creating a negative feedback loop. Thus, how S6K1 impacts HSC ageing and stem cell functions remains an enigma. We have assessed the role of S6K1 in HSC development and function under steady-state as well as during recovery of hematopoietic system following myelosuppressive stress. We used a genetic model of S6K1 knockout mice (S6K1-/-). S6K1 deficiency in bone marrow hematopoietic cells resulted in decrease of absolute number of bone marrow hematopoietic progenitor cells as well as HSCs (Lin- Sca1+ c-Kit+; LSK) were significantly reduced relative to controls (n=14 in each group, p Disclosures No relevant conflicts of interest to declare.

Abstract 810 Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in KIT involving an aspartic acid to valine substitution is found in ∼90% of patients with systemic mastocytosis (SM) and in ∼40% of core binding factor acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. In mice, presence of this mutation is sufficient to recapitulate many cardinal features of human SM. This mutation changes the conformation of KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth, which is resistant to imatinib and shows little therapeutic efficacy in response to Dasatinib in most SM patients. As there are currently no efficacious therapeutic agents against this mutation, we sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells (HSC/Ps) in diseases such as AML and SM. Previously, we and others have demonstrated that the regulatory subunit of PI3K, p85α, is required for KITD814V (murine homolog) induced transformation. Although difficult to target, we hypothesized that perhaps the downstream effectors of the PI3K signaling pathway, in particular p21 activated kinase (PAK1) and its upstream effectors including guanine exchange factors (GEF) such as Tiam1, Trio and Vav as well as the Rho family of GTPases (Rac) contribute to gain-of-function mutant-mediated transformation. We show that KITD814V (mouse) and KITD816V (human) bearing leukemic cells exhibit constitutive activation of PAK, Rac GTPases, and GEF Vav. Importantly, treatment of KITD814V bearing murine cells or mastocytosis patient derived cells bearing the KITD816V mutation with an allosteric inhibitor of PAK1 (i.e. IPA-3) results in significant inhibition in growth due to enhanced apoptosis. Consistently, expression of a dominant negative form of PAK1 (K299R) in KITD814V bearing cells profoundly inhibited their growth but not the growth of normal cells. Upstream of PAK, we show that suppression of Rac GTPases by expression of a dominant negative form of Rac (RacN17) abrogates activating KIT-induced hyperproliferation, and activity of downstream effector, PAK1. Although both Rac1 and Rac2 are activated due to the presence of KITD814V in primary HSC/Ps; loss of Rac1 only modestly corrects the growth of KITD814V bearing cells and loss of Rac2 contributes to only 50% correction. In contrast, loss of both Rac1 and Rac2 in HSC/Ps resulted in 75% correction in KITD814V induced ligand independent growth in vitro. In vivo, Rac repression significantly delayed the onset of KITD814V induced myeloproliferative neoplasms (MPN). Although, all KITD814V bearing mice died around 20 days of transplantation due to splenomegaly, increased white cell counts and massive lung infiltration by leukemic cells; KITD814V bearing mice in which Rac was repressed showed prolonged survival, significantly reducted spleen size, white cell counts and myeloid cell infiltration in the lungs. Prior studies have shown that Rac GTPases can be activated by GEFs such as Tiam1, Trio and Vav. To assess the specific role of these GEFs in KITD814V induced transformation, we utilized small molecule inhibitors that uniquely target different GEFs. We synthesized and utilized a novel inhibitor of Rac, EHop-016, which is based on the structure of an existing GEF inhibitor, NSC23766. While NSC23766 targets Tiam1 and Trio, EHop-016 targets Vav. The IC50 of EHop-016 is ∼50 fold lower than that of NSC23766. Using these two drugs, we demonstrate that EHop-016 is 50-fold more potent in inhibiting the growth of both murine and human patient derived leukemic cells compared to NSC23766. These observations were confirmed utilizing mice and bone marrow cells deficient in the expression of Vav1 engineered to express the KITD814V mutation. Taken together, a series of experiments using knockout mouse models, mouse models of MPN, dominant negative approaches, and a novel allosteric inhibitor of PAK1 and a novel small molecule inhibitor of GEF Vav provide a mechanism of KITD816V induced transformation and provide potential novel therapeutic targets for treating oncogenic KIT bearing neoplasms. Disclosures: No relevant conflicts of interest to declare.

Abstract 916 Erythropoiesis is a dynamic, multistep process in which hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Under normal physiologic condition, erythropoiesis takes place primarily in the bone marrow of humans and mice. Efficient stress erythropoiesis is crucial to survival and recovery from various pathophysiologic conditions including blood loss, anemia, and therapeutic procedures used in the treatment of hematological malignancies such as chemotherapy and stem cell transplantation. Impaired stress erythropoiesis can be fatal under these conditions. In order to develop improved strategies to stimulate stress erythropoiesis in patients with such conditions, it is critical to identify the molecular mechanisms that regulate this process. While many of the downstream signaling molecules and pathways have been elucidated in steady state erythroid cell development, the major regulators under stress conditions remain to be defined. The small families of Rho GTPases and their downstream effectors, Rho kinases have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells and inflammatory cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases, and their physiologic role in erythropoiesis is not known. Utilizing mice deficient in the expression of ROCK1, we demonstrate no significant difference with respect to erythroid parameters in peripheral blood under steady-state conditions (n=19 mice for each genotype). The frequency of Ter119+, CD71+ or double-positive erythroid cells in the bone marrow and spleen of ROCK1-deficient mice was comparable to wild type (WT) controls at basal levels (n=7). In response to myelotoxic stress, ROCK1 deficient Ter119 positive cells demonstrated enhanced survival and recovery compared to WT controls (n=3–6, *p< 0.05). Further, using a phenylhydrazine (PHZ)-induced murine model of hemolytic anemia, we demonstrate that ROCK1-deficient mice exhibit increased red blood cells and enhanced hematocrits relative to WT mice (n=6, *p< 0.05). In addition, ROCK1−/− mice support efficient erythro-splenomegaly, and exhibit a threefold increase in splenic weight after PHZ injection compared to WT controls (n=6, *p< 0.05). Flow cytometric analysis revealed increased frequency of Ter119/CD71 double positive erythroid progenitor pool in ROCK1−/− spleens after PHZ treatment compared to WT (n=6, *p< 0.05). Furthermore, histopathological analysis of WT and ROCK1−/− spleens following PHZ treatment revealed a defined population of white pulp and red pulp in control spleens but enhanced extramedullary erythropoiesis in ROCK1−/− spleens (n=3, *p< 0.05). In vitro colony forming assays showed that ROCK1-deficient splenocytes generated more erythroid colony forming units (CFU-Es) and eventually generated more erythroid-burst forming units (BFU-Es) compared to WT splenocytes in response to different concentrations of EPO and combination of EPO and SCF (n=3, *p< 0.05). Deficiency of ROCK1 also resulted in enhanced survival of mice treated with PHZ compared to controls (n=17 mice per group, *p< 0.05). The enhanced survival of ROCK1-deficient mice in response to PHZ was associated with reduced reactive oxygen species (ROS) levels compared to WT (n=6 mice per group, *p< 0.05). Bone marrow transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous (n=6 mice per genotype, *p< 0.05). Remarkably, the red cell phenotype observed in ROCK1−/− mice is similar to that reported in mice deficient in p53. We show that ROCK1 binds p53 directly and regulates its stability and expression (n=3). In absence of ROCK1, p53 phosphorylation and expression is reduced in ROCK1−/− erythroblasts (n=3). Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 868 Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), and acute myelogenous leukemia (AML). An activating KIT receptor mutation of aspartic acid to valine at codon 814 in mice (KITD814V) or codon 816 in humans (KITD816V) results in altered substrate recognition and constitutive tyrosine autophosphorylation leading to promiscuous signaling. Consequently, cells bearing oncogenic form of KIT (KITD814V) demonstrate ligand independent proliferation in vitro and MPD in vivo. However, the intracellular signals that contribute to KITD814V induced MPD are not known. Here, we show the constitutive phosphorylation of SHP2 in cells bearing KITD814V, but not WT KIT, which was inhibited by treatment with a novel SHP2 inhibitor, II-B08 (*p < 0.05). In addition, treatment with II-B08 suppressed the growth of cells bearing KITD814V, but not WT KIT (*p < 0.05), Human mast cell line HMC1.2 (*p < 0.05), and Human CD34+ cells bearing KITD816V (*p < 0.05). Likewise, deficiency of SHP2 in primary bone marrow cells resulted in a significant repression in constitutive growth of cells bearing KITD814V (*p < 0.05). To determine the mechanism behind the repression in ligand independent growth of cells bearing KITD814V by II-B08, we examined the role of SHP2 in cell survival. We observed a dose dependent increase in apoptosis of cells bearing KITD814V compared to WT KIT in the presence of II-B08 (*p < 0.05). Similarly, deficiency of SHP2 resulted in increased apoptosis of cells bearing KITD814V (*p < 0.05). In an effort to identify the mechanism behind reduced growth and increased apoptosis of cells bearing KITD814V in the presence of II-B08, we examined whether SHP2 regulates the activation of AKT in KITD814V bearing cells. We found constitutive activation of AKT in cells bearing KITD814V, but not WT KIT, which was significantly inhibited upon II-B08 treatment suggesting that SHP2 regulates ligand independent growth and survival of KITD814V bearing cells in part by regulating the activation of AKT. To further determine the signaling molecules that co-operate with SHP2 in KITD814V induced MPD, we examined proteins that potentially interact with SHP2 in KITD814V bearing cells and whether inhibition of SHP2 activity by II-B08 suppresses these co-operating protein interactions. SHP2 constitutively bound to p85α and Gab2 in cells bearing KITD814V, but not in WT KIT bearing cells, which was inhibited upon II-B08 treatment. To further assess whether the binding of SHP2 to p85α, Gab2 and KITD814V is sufficient to induce MPD, we generated a KIT mutant receptor, KITD814V-F7, in which seven tyrosine residues in KITD814V (known to bind SH2 containing proteins at tyrosine 567, 569, 702, 719, 728, 745, and 934) were converted to phenylalanine, and KITD814V-Y719, in which only tyrosine residue 719 (binding site for p85α) was added back to the KITD814V-F7 receptor. We observed constitutive binding of SHP2 and Gab2 to p85α in cells bearing KITD814V and KITD814V-Y719, but not in WT KIT or KITD814V-F7 bearing cells. In addition, conversion of all the seven intracellular tyrosine residues in KITD814V to phenylalanine (KITD814V-F7) resulted in complete loss of ligand independent growth in vitro (*p < 0.05) and significantly delayed the progression of MPD in vivo (*p < 0.05). Importantly, restoration of tyrosine residue at position 719 (KITD814V-Y719) was sufficient to induce ligand independent growth in vitro and MPD in vivo to KITD814V levels. Furthermore, deficiency of Gab2 resulted in significant repression in constitutive growth of cells bearing KITD814V (*p < 0.05). These results demonstrate that p85α recruits SHP2 and Gab2 to KITD814V at Y719, which might contribute to KITD814V induced MPD. Moreover, II-B08 enhances the efficacy of PI3Kinase inhibitor LY294002 in suppressing KITD814V induced ligand independent growth in vitro (*p < 0.05) and MPD in vivo (*p < 0.05). Taken together our results demonstrate that SHP2 is a druggable target which cooperates with PI3Kinase in inducing MPD. Disclosures: No relevant conflicts of interest to declare.

Abstract 1435 Poster Board I-458 Gain-of-function mutations of the receptor tyrosine kinase KIT have been associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myelogenous leukemia (AML). A mutation of aspartic acid to valine (KITD814V) in the activation loop of KIT results in altered substrate recognition and constitutive tyrosine autophosphorylation. However, the intracellular mechanisms that contribute to promiscuous signaling via this mutation are poorly understood. We have previously shown that KITD814V is sufficient to induce ligand independent growth in primary hematopoietic stem and progenitor cells (HSC/P) in vitro as well as transformation in vivo. However, it is not known whether ligand induced (i.e. KITL) signals in vivo contribute to transformation. To assess this, we generated a chimeric receptor (CHR) in which the extracellular domain of KIT was replaced with the human-macrophage colony stimulating factor receptor (h-MCSFR) to inhibit endogenous binding of murine SCF. In vitro thymidine incorporation assay confirmed that the WTCHR is only functionally responsive to human (h)-MCSF stimulation, but not to murine (m)SCF nor murine MCSF stimulation, and that CHRD814V receptor maintains ligand independent growth potential similar to the KITD814V receptor. To determine if the myeloproliferative disease (MPD) could occur in the absence of endogenous ligand binding, a murine transplantation model was utilized to compare CHRD814V and KITD814V-induced transformation. In vivo results demonstrated no significant difference in the onset of MPD in mice bearing KITD814V vs. CHRD814V (median survival 52 days vs. 49 days, n=10 to 20 mice), with similar disease manifestation in the two groups including splenomegaly, hepatomegaly, myeloid cell infiltration and elevated white blood cell counts (n=10 to 20). Transforming potential in the absence of KIT extracellular domain strongly suggested that self-association of KITD814V was sufficient to induce transformation rather than influences from endogenous ligand. To further assess the relative contribution of intracellular tyrosine residues in transformation, a CHRD814V mutant was generated in which seven critical tyrosine residues including the binding sites for Src family kinases, Grb2, p85α regulatory subunit of class IA PI3Kinase, PLC-g, Ras-GAP, and Grb7 were mutated to phenylalanines (i.e.CHRD814V-F7). Our results show that CHRD814V-F7 bearing primary bone marrow cells lost ligand independent growth potential in vitro, suggesting that seven tyrosine induced signaling may play a critical role in KITD814V-induced ligand independent transformation. Consistently, transplantation studies demonstrated that mice bearing bone marrow cells expressing CHRD814V-F7 exhibited a notable delay in MPD development (median survival= 95 days, n=4 to 8). These results suggest that intracellular tyrosines are crucial for ligand independent proliferation and transformation. Next, we individually restored each of the seven tyrosine residues back into CHRD814V-F7 to determine the importance of pathways essential for ligand independent growth and transformation. We found that among the single tyrosine add back receptors, only restoring p85α subunit binding site (Y719) alone was sufficient to induce promiscuous cellular growth, proliferation, and survival. Transplantation studies revealed that restoration of Y719 was sufficient to induce transformation in vivo (median survival= 55 days, n=5). Furthermore, restoration of other individual tyrosine residues demonstrated a notable delay in CHRD814V-induced transformation in vivo (median survival= 83∼120 days, n=5 to 12) and a minimal contribution to ligand independent growth in vitro. Taken together, our results demonstrate: 1) KITD814V induced transformation in vivo occurs in the absence of ligand stimulation; 2) intracellular tyrosine residues play an essential role in KITD814V induced transformation in vitro and in vivo and; 3) of all the critical tyrosine residues present in KIT, presence of tyrosine at position 719 is sufficient to completely restore ligand independent growth in vitro and transformation in vivo. Disclosures No relevant conflicts of interest to declare.

SummaryWe show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3, and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene-bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient-derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth, and loss of actin polymerization in oncogene-bearing cells leading to significantly prolonged life span of leukemic mice. In summary we describe a pathway involving PI3K/Rho/ROCK/MLC that may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans.