*° contributed equally to this work. Introduction: Systemic Mastocytosis (SM) is a heterogeneous disorder characterized by mast cells (MCs) accumulation in various tissues and associated with KIT mutations (KIT D816V) in more than 90% of the cases. It includes indolent (ISM) and advanced diseases (advSM), which are associated with additional molecular abnormalities. For advSM, recent clinical studies have shown that Midostaurin, a kinase inhibitor of WT and mutant KIT, induces high rate of responses associated with significant improvement of prognosis. However, complete responses are infrequent and relapses occur in a significant proportion of patients. Therefore, combination therapies are needed to increase overall response rate and prevent relapses. Venetoclax is a selective orally bioavailable BCL-2 inhibitor that induces cell death and is currently used for treatment of various lymphoid and myeloid malignancies. In an attempt to identify novel diagnostic and prognostic markers and potentially new therapeutic targets for mastocytosis, bone marrow sections of patients with different categories of mastocytosis were analyzed by IHC using anti-BCL-2 antibodies. BH-3 profiling was used to assess BH-3 proteins dependency, and sensitivity to Venetoclax alone or in combination of Midostaurin. Methods: Thirty-three adult patients were included in this preliminary study. According to the WHO classification, patients were classified as having ISM (n=10), Smoldering SM (SSM n=1), advSM (n=16, including SM-AHN (n=9), MC leukemia (MCL n=4), MC sarcoma (MCS n=2)). Most patients were KIT D816V (n=30; 90.9%); two MCL and one MCS exhibited extracellular and juxtamembrane mutations, respectively. Among these patients, 9 were treated with Midostaurin as first line therapy. Formalin fixed bone marrow sections were performed at diagnosis and during follow up. Mast cells were identified by Giemsa staining and as CD117 and tryptase positive cells. BCL-2 staining was performed by immunohistochemistry in formalin paraffin embedded fixed section. BCL-2 staining was considered as positive (>5%), heterogeneous (partial staining) or homogeneous (>80% positive cells), of high or low intensity (> or = or < to residual T cells). BH3 profiling was performed in ROSA KIT WT and ROSA KIT D816V using Cytochrome C upon exposure to distinct BH3 peptides/mimetics. Results: In ISM, BCL-2 staining was negative (n= 2/10) or when positive only in rare MCs (n=8/10), with low intensity. In contrast, all advSM cases were positive (16/16) with high (13/16), and homogeneous (6/16) staining. In MCL and MCS, BCL-2 staining was always positive with a homogeneous and high staining. In patients treated with Midostaurin, BCL-2 staining was performed before and three months after treatment initiation. Although MCs infiltration was reduced at least by 50% in all cases, number of BCL-2 positive cells and intensity of staining remain unchanged. In vitro, flow cytometry analysis showed that both MCL-like cell lines (ROSA KIT WT and ROSA KIT D816V) expressed BCL-2, MCL-1 and BCL-XL proteins. When treated with Midostaurin (200nM) for 48 hours, expression of BCL-XL and MCL-1 significantly decreased in MC lines especially the one with KIT D816V mutation. Interestingly, BCL-2 expression remained unchanged upon Midostaurin treatment, which was consistent with in vivo observations. Dynamic profiling performed in ROSA cell lines revealed that priming by midostaurin dramatically enhanced apoptotic dependencies to BCL-2 and other BH-3 proteins (>20% of apoptosis), especially in ROSA KIT D816V (figure). Conclusion: High expression of BCL-2 is associated with advSM and may participate to the pathogenesis of the disease, to its resistance to conventional chemotherapies and to partial resistance to Midostaurin. Consistent with its effect in reducing MCL-1 and BCL-XL expression, Midostaurin restored apoptotic dependency to BCL2 in human MCL-like cells, thereby suggesting that midostaurin could sensitize mast cell tumor to venetoclax. Our results provide thus a rationale to use a combination of Midostaurin and Venetoclax to treat AdvSM patients. Figure Disclosures Dubreuil: AB Science: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hermine:AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. OffLabel Disclosure: Venetoclax preclinical studioes on mastocytosis

Hematopoiesis, a process that results in the differentiation of all blood lineages, is essential throughout life. The production of 1x1012 blood cells per day, including 200x109 erythrocytes, is highly dependent on nutrient consumption. Notably though, the relative requirements for micronutrients during the perinatal period, a critical developmental window for immune cell and erythrocyte differentiation, have not been extensively studied. More specifically, the impact of the vitamin C/ascorbate micronutrient on perinatal as compared to adult hematopoiesis has been difficult to assess in animal models. Even though humans cannot synthesize ascorbate, due to a pseudogenization of the L-gulono-γ-lactone oxidase (GULO) gene, its generation from glucose is an ancestral mammalian trait. Taking advantage of a Gulo-/- mouse model, we show that ascorbic acid deficiency profoundly impacts perinatal hematopoiesis, resulting in a hypocellular bone marrow (BM) with a significant reduction in hematopoietic stem cells, multipotent progenitors, and hematopoietic progenitors. Furthermore, myeloid progenitors exhibited differential sensitivity to vitamin C levels; common myeloid progenitors and megakaryocyte-erythrocyte progenitors were markedly reduced in Gulo-/- pups following vitamin C depletion in the dams, whereas granulocyte-myeloid progenitors were spared, and their frequency was even augmented. Notably, hematopoietic cell subsets were rescued by vitamin C repletion. Consistent with these data, peripheral myeloid cells were maintained in ascorbate-deficient Gulo-/- pups while other lineage-committed hematopoietic cells were decreased. A reduction in B cell numbers was associated with a significantly reduced humoral immune response in ascorbate-depleted Gulo-/- pups but not adult mice. Erythropoiesis was particularly sensitive to vitamin C deprivation during both the perinatal and adult periods, with ascorbate-deficient Gulo-/- pups as well as adult mice exhibiting compensatory splenic differentiation. Furthermore, in the pathological context of hemolytic anemia, vitamin C-deficient adult Gulo-/- mice were not able to sufficiently increase their erythropoietic activity, resulting in a sustained anemia. Thus, vitamin C plays a pivotal role in the maintenance and differentiation of hematopoietic progenitors during the neonatal period and is required throughout life to sustain erythroid differentiation under stress conditions.

Purpose: Succinate dehydrogenase deficient (dSDH) tumors, including pheochromocytoma/paraganglioma, hereditary leiomyomatosis and renal cell cancer-associated renal cell carcinoma (HLRCC-RCC), and gastrointestinal stromal tumors (GISTs) without KIT or platelet derived growth factor receptor alpha mutations are often resistant to cytotoxic chemotherapy, radiotherapy, and many targeted therapies. We evaluated guadecitabine, a dinucleotide containing the DNA methyltransferase inhibitor decitabine, in these patient populations. Patients and Methods: Phase II study of guadecitabine (subcutaneously, 45mg/m2/day for 5 consecutive days, planned 28-day cycle) to assess clinical activity (according to Response Evaluation Criteria in Solid Tumors v.1.1) across 3 strata of patients with dSDH GIST, pheochromocytoma/paraganglioma, or HLRCC-RCC. A Simon optimal two-stage design (target response rate 30% rule out 5%) was used. Biologic correlates (methylation and metabolites) from peripheral blood mononuclear cells (PBMCs), serum, and urine were analyzed. Results: Nine patients (7 with dSDH GIST, 1 each with paraganglioma and HLRCC-RCC, 6 females and 3 males, age range 18-57 years-old) were enrolled. Two patients developed treatment-limiting neutropenia. No partial or complete responses were observed (range 1-17 cycles of therapy). Biologic activity assessed as global demethylation in PBMCs was observed. No clear changes in metabolite concentrations were observed. Conclusions: Guadecitabine was tolerated in patients with dSDH tumors with manageable toxicity. While 4/9 patients had prolonged stable disease there were no objective responses. Thus, guadecitabine did not meet the target of 30% response rate across dSDH tumors at this dose, though signs of biologic activity were noted.