Investigating the clonal and biological underpinnings of systemic mastocytosis with an associated hematological neoplasm

Abstract

Abstract Systemic mastocytosis (SM) is a disease characterized by the clonal expansion of abnormal mast cells driven by KIT D816V activatingmutations in about 95% of cases. Interestingly, in some patients with fulminant SM symptomatology, KIT mutations are only detected by ddPCR at variant allele frequencies (VAFs) of <2% in the bone marrow (BM). SM with an associated hematological neoplasm (SM-AHN) is the commonest advanced SM and chronic myelomonocytic leukemia (CMML) is the most frequent AHN diagnosed. Despite the frequent co-occurrence of CMML and SM, two critical knowledge gaps remain: (1) the clonal architecture of SM-AHN is poorly understood and (2) no studies have investigated the biological underpinnings that explain the high frequency of synchronous SM and CMML. To address these gaps we performed single-cell DNA-seq and surface protein assessment of BM mononuclear cells from 7 SM-AHN samples. Using a manual cell annotation strategy we were able to approximate both clinically annotated numbers of mast cells and KIT VAFs suggesting that cryopreserved specimens recapitulate clinically reported morphologic and molecular features. Next, we observed that while expansion of mast cells was observed in all cases, up to 96% of these mast cells were KIT wildtype. This provides an explanation for the observation that low VAF KIT mutant SM-AHN can be highly symptomatic likely through recruitment and activation of wildtype mast cells. We also observed that even in very low VAF KIT D816V cases, KIT mutations were always detectable in hematopoietic stem and progenitor cells (HSPCs). Moreover, KIT D816V mutated HSPCs were universally derived from ancestral clones that were co-mutated with AHN mutations suggesting that even low VAF KIT mutant SM and AHN are of common clonal origin. To further support the latter conclusion, we leveraged our CMML patient derived xenograft (PDX) bank to identify cases with KIT mutation but no overt SM diagnosis. We were able to observe human mast cell aggregates positive for CD117, CD25 and mast cell tryptase by immunohistochemistry in the PDX bone marrow suggestive of SM-AHN and supporting the common clonal origin of this disease. To investigate the cell extrinsic association between CMML and SM, we developed a heterotopic in vivo model that injected the HMC 1.2 mast cell line and one of two CMML-like cell lines THP-1 and U937 in the contralateral flanks of NSG mice. We observed that compared to controls with solitary HMC 1.2 or monocytic leukemia tumors, mast cell tumors grew larger with a concomitant monocytic tumor. Conversely, monocytic tumors with a concomitant mast cell tumor demonstrated decreased growth compared to monocytic tumors only (fold change bilateral vs single: THP-1=0.56 p=0.049, HMC 1.2 [THP-1]=1.60 p=0.01 n=8 per group; U937=0.47 p=0.0008, HMC 1.2 [U937]=1.99 p=0.02 n=11 per group). This observation was recapitulated in in vitro models as well as a model in which the HMC 1.2 cell line was heterotopically transplanted in combination with orthotopically transplanted monocytic cells and appropriate controls. In this model, death from monocytic leukemia was improved from 40 days to 46 days (p=0.01 n=8 per group). Collectively, these data suggest that KIT mutant mast cells reproducibly increase proliferative capacity while monocytic leukemia cells decrease proliferative capacity when both are present. Given these findings, we reasoned that our model could provide a platform to understand KIT inhibitor resistance mechanisms in SM-AHN for two reasons: (1) Avapritinib is a potent and selective KIT inhibitor that effectively suppresses mast cells and (2) 78% to 86% of SM-AHN progression events are due to AHN progression. Therefore we hypothesized that the use of Avapritinib in our heterotopic model would lead to potent inhibition of mast cell tumors and a subsequent loss of monocytic proliferative suppression. Indeed treatment with 30mg/kg for 6 days resulted in a rescue of monocytic leukemia growth in the Avapritinib treated group supporting the notion that AHN progression is likely due to the loss of mast-cell derived secreted factors which suppress monocytic leukemia (fold change bilateral vs single: U937 vehicle=0.39 p=0.009, U937 Avapritinib=0.92 p=0.75 n=5 per group). RNA-sequencing of tumors, broad cytokine profiling, and phosphoproteomics are underway to identify networks that are responsible for our observed phenotype and may be leveraged as therapy for both CMML and SM-AHN.