Abstract: The KIT receptor tyrosine kinase has important roles in hematopoiesis. We have recently produced a mouse model for imatinib resistant gastrointestinal stromal tumor (GIST) carrying the KitV558Δ and KitT669I (human KITT670I) mutations found in imatinib-resistant GIST. The KitV558Δ;T669I/+ mice developed microcytic erythrocytosis with an increase in erythroid progenitor numbers, a phenotype previously seen only in mouse models of polycythemia vera with alterations in Epo or Jak2. Significantly, the increased hematocrit observed in KitV558Δ;T669I/+ mice normalized upon splenectomy. In accordance with increased erythroid progenitors, myeloerythroid progenitor numbers were also elevated in the KitV558Δ;T669I/+ mice. Hematopoietic stem cell (HSC) numbers in the bone marrow (BM) of KitV558Δ;T669I/+ mice were unchanged in comparison to wild-type mice. However, increased HSC numbers were observed in fetal livers and the spleen and peripheral blood of adult KitV558Δ;T669I/+ mice. Importantly, HSC from KitV558Δ;T669I/+ BM had a competitive advantage over wild-type HSC. In response to 5-fluorouracil treatment, elevated numbers of dividing Lin−Sca+ cells were found in the KitV558Δ;T669I/+ BM compared to wild type. Our study demonstrates that signaling from the KitV558Δ;T669I/+ receptor has important consequences in hematopoiesis enhancing HSC self-renewal and resulting in increased erythropoiesis.

Abstract: Deciphering Multiple Myeloma evolution in the whole bone marrow is key to inform curative strategies. Here, we perform spatial-longitudinal whole-exome sequencing, including 140 samples collected from 24 Multiple Myeloma patients during up to 14 years. Applying imaging-guided sampling we observe three evolutionary patterns, including relapse driven by a single-cell expansion, competing/co-existing sub-clones, and unique sub-clones at distinct locations. While we do not find the unique relapse sub-clone in the baseline focal lesion(s), we show a close phylogenetic relationship between baseline focal lesions and relapse disease, highlighting focal lesions as hotspots of tumor evolution. In patients with ≥3 focal lesions on positron-emission-tomography at diagnosis, relapse is driven by multiple distinct sub-clones, whereas in other patients, a single-cell expansion is typically seen ( p   〈  0.01). Notably, we observe resistant sub-clones that can be hidden over years, suggesting that a prerequisite for curative therapies would be to overcome not only tumor heterogeneity but also dormancy.

Abstract: Smoldering myeloma (SMM) is associated with a high-risk of progression to myeloma (MM). We report the results of a study of 82 patients with both targeted sequencing that included a capture of the immunoglobulin and MYC regions. By comparing these results to newly diagnosed myeloma (MM) we show fewer NRAS and FAM46C mutations together with fewer adverse translocations, del(1p), del(14q), del(16q), and del(17p) in SMM consistent with their role as drivers of the transition to MM. KRAS mutations are associated with a shorter time to progression (HR 3.5 (1.5–8.1), p  = 0.001). In an analysis of change in clonal structure over time we studied 53 samples from nine patients at multiple time points. Branching evolutionary patterns, novel mutations, biallelic hits in crucial tumour suppressor genes, and segmental copy number changes are key mechanisms underlying the transition to MM, which can precede progression and be used to guide early intervention strategies.

Abstract: We previously produced a mouse model for GIST by introducing a Kit -activating mutation ( Kit V558Δ ) originally identified in a case of familial GIST into the mouse Kit gene ( 4 ). Here we describe the generation of a mouse model for imatinib-resistant GIST that includes both the Kit V558Δ mutation and a secondary Kit T669I “gatekeeper” mutation, as a tool for developing therapeutic strategies for imatinib-resistant GIST and for investigating the consequences of KIT oncogenic signaling in other KIT-dependent cell systems, in particular in hematopoiesis. We found that Kit V558Δ;T669I/+ mice exhibited increased ICC hyperplasia and more pronounced (mast cells) as well as distinct hematopoietic phenotypes (microcytic erythrocytosis) as compared with mice carrying only the primary Kit mutation ( Kit V558Δ/+ ) ( Table P1 ). Importantly, although GIST lesions of Kit V558∆/+ and Kit V558∆;T669I/+ mice were similar in histology and oncogenic signaling, the Kit V558∆;T669I/+ mice were resistant to imatinib and to dasatinib tyrosine kinase-inhibitor therapy. This resistance could be overcome by treatment with the tyrosine kinase inhibitors sunitinib and sorafenib, supporting a rationale for using sunitinib as second-line therapy for imatinib-refractory GIST ( Table P1 ). Interestingly, in the erythroid lineage, a polycythemia vera-like phenotype, with highly increased microcytic red blood cell numbers, was observed in Kit V558Δ;T669I/+ mice. In agreement with our findings, the activity of the KIT V558Δ;T669I kinase in vitro is nearly doubled ( 5 ), possibly explaining the KIT hyperactivity observed in the ICC, mast cell, and erythrocyte lineages in Kit V558Δ;T669I/+ mice. Our results highlight the importance of a combination of factors, including the type of activation-mutation and cellular context, involved in determining mutant/oncogenic phenotypes in vivo . In summary, the Kit V558∆;T669I/+ mice provide an excellent tool for developing therapeutic strategies for imatinib-resistant GIST and for investigating the consequences of KIT oncogenic signaling in other KIT-dependent cell systems, particularly pigment development (melanogenesis) and blood cell development (hematopoiesis). Kit originally was discovered as an oncogene of a feline sarcoma virus ( 2 ). Later, gain-of-function mutations in the KIT receptor were reported in human mastocytosis, seminoma, and a subset of acute myelogenous leukemia, and in 1998, Kit mutations were also identified in GIST ( 3 ). GIST is the most common mesenchymal tumor of the gastrointestinal tract. GISTs express KIT, and they presumably derive from KIT-expressing ICC progenitors or from ICCs. Today, we know that the principal genetic events responsible for the pathogenesis of GIST are gain-of-function mutations in the Kit gene, found predominantly in the juxtamembrane domain of the KIT receptor, which disrupt the conformational integrity of the protein and thus diminish the autoinhibitory function of the juxtamembrane domain. Mutations in the extracellular and kinase domains of KIT also have been described. Imatinib therapy is effective in GISTs with Kit -activating mutations in the juxtamembrane domain, some kinase-domain mutations, and extracellular domain mutations but is ineffective in tumors with Kit mutations that stabilize the active conformation of the kinase. The KIT receptor tyrosine kinase and its cognate ligand, Kit ligand, have critical functions in several cell lineages during embryonic development and in adult mammals. In hematopoiesis, KIT function is critical in the stem cell hierarchy and the development of red blood cells (erythropoiesis), mast cells, and platelets. A hallmark of mice carrying Kit loss-of-function mutations is a profound decrease in erythropoiesis (macrocytic anemia) and mast cell development. In addition, Kit is involved in several stages of melanogenesis and in primordial germ cells, spermatogenesis, and oogenesis. A major KIT-expressing cell in the gastrointestinal tract is the interstitial cell of Cajal (ICC). ICCs function as pacemakers of gut motility. In an elegant and pivotal study in 1992, Nishikawa and colleagues ( 1 ) showed that an antagonistic KIT antibody could interfere with autonomous gut movement in mice, essentially giving rise to a disorder resulting in the paralysis of the bowel, a condition known as “lethal paralytic ileus,” and it was shown that mice with Kit loss-of-function mutations have impaired pacemaker activity and lack ICC networks. Targeted drug therapy enables the specific inhibition of enzymes involved in the pathogenesis of cancer. Protein kinases are critical regulators of cellular processes, including cell proliferation and cell survival. Oncogenic driver mutations are highly prevalent in protein kinases; therefore, protein kinases are logical targets for therapeutic intervention. Although the design of kinase inhibitors that display high degrees of specificity has met with many challenges, the prospect of their utility in reducing side effects commonly associated with standard chemotherapy continues to be a driving force behind their development. The first breakthrough drug to come out of this effort was imatinib, an inhibitor of the KIT and PDGF receptor tyrosine kinases and the BCR-ABL fusion protein, proteins involved in the development of several cancers, including gastrointestinal stromal tumor (GIST) and chronic myelogenous leukemia. Gain-of-function mutations in the KIT receptor are a hallmark of GIST. Today imatinib treatment is standard therapy for patients with GIST. Unfortunately, long-term treatment of GIST with imatinib is associated with the development of drug resistance, often as the result of the acquisition of a secondary mutation in the kinase domain of the KIT receptor. Given the clinical importance of imatinib resistance, the development of new strategies for the treatment of GIST is very important. In the current study, we have developed a mouse model for imatinib-resistant GIST.

Abstract: 8026 Background: PARP inhibitors can induce synthetic lethality in tumors characterized by homologous recombination deficiency (HRD). Two PARP inhibitors are approved for the treatment of BRCA mutated ovarian cancer. HRD can be detected by evaluating genome-wide loss of heterozygosity (LOH), which is associated with response to PARP inhibition. Myeloma (MM) is a genetically unstable tumor and we hypothesized that LOH could be detected in patient samples, supporting a potential role for PARP inhibition in MM. Methods: We analyzed 406 cases at all disease stages: MGUS (n = 7), smoldering MM (SMM, n = 30) newly diagnosed MM (NDMM, n = 71), treated MM (TRMM, n = 64) and relapsed MM (RLMM, n = 234). CD138+ plasma cell DNA underwent targeted next generation sequencing (FoundationOne Heme) interrogating 405 cancer related genes and 3543 single nucleotide polymorphisms (SNPs) across the genome. An algorithm using the minor allele frequencies of the examined SNPs and copy number profile across the 22 autosomal chromosomes identified LOH segments. Events unlikely to be caused by HRD, e.g. whole chromosome or chromosome-arm loss, were excluded. The percentage of genomic LOH for each sample was calculated as the sum of the lengths of included LOH segments divided by the length of the interrogated genome. Results: We found evidence of HRD detected by LOH with higher LOH selecting for patients with poor outcome. LOH increases with advancing disease stage from a median of 0.3% in MGUS to 3.1% in RLMM. LOH was highest in the PR and MMSET molecular subgroups and correlated significantly with the gene expression defined risk score GEP70 (R = 0.4, p 〈 0.001) and proliferation index (R = 0.4, p 〈 0.001). Outcome of RLMM patients, the biggest clinical group, was analyzed and patients with LOH above the 3 rd quartile (≥5%LOH) had significantly worse overall survival than those with lower levels (p 〈 0.001). LOH correlates and overlaps with other metrics of poor prognosis, suggesting it may be a prognostic marker itself. Conclusions: We demonstrated LOH in MM samples, increasing as disease progresses and associated with poor prognosis. These data support the further evaluation of PARP inhibitors in MM patients, particularly in the relapsed setting with a high unmet need for new treatments.

Abstract: Introduction: The proto-oncogene MYC (locus 8q24.21) is a key transcription factor in multiple myeloma (MM) resulting in significant gene deregulation and impacting on many biological functions, including cell growth, proliferation, apoptosis, differentiation, and transformation. Chromosomal rearrangement and copy number change at the MYC locus are secondary events involved in MM progression, which are thought to lead to aggressive disease. Current analyses of the MYC locus have not been large and have reported rearrangements in 15% of new-diagnosed MM. However, more recent studies using advanced genomic techniques suggest that the frequency of MYC rearrangements may be much higher, and that a full reassessment of the role of MYC in MM pathogenesis may be critical. In this study, we analyzed 1280 MM patients to provide a better understanding of the role of this important genomic driver in MM pathogenesis. Methods: In total, 1280 tumor normal pairs of CD138 sorted bone marrow plasma cells and their germline control samples were analyzed by: 1. Targeted sequencing of 131 genes and 27 chromosome regions (n=100) with 4.5 Mb captured region surrounding MYC ; 2. Exome sequencing (n=461) with 2.3 Mb captured region surrounding MYC ; 3. Whole genome sequencing (n=719). Normalized tumor/germline depth ratio in targeted-sequencing cases and MANTA were used for detection of somatic copy number and structural variants. Expression analysis was performed using RNA-seq or microarrays. Results: MYC translocations were found in 25% (323/1280) of patients and occurred most frequently as inter-chromosomal translocations involving 2-5 chromosomes (90%, 291/323). Of the remaining cases, 5% (17/323) of the translocations involved inversion of chromosome 8 and 5% (15/323) were complex, affecting more than 5 chromosomal loci. The proportion of MYC translocations involving 2, 3, 4, and 5 loci was 62% (200/323), 23% (74/323), 8% (26/323) and 3% (8/323), respectively. Using abnormal rearranged cases (29/100), we found copy number imbalances & gt;14.2 kb in size associated with a MYC translocation in 76% (22/29). Another 7% (2/29) of cases with translocations showed complex intra-chromosomal rearrangement. A region of 2.0 Mb surrounding MYC was identified as a translocation breakpoint hot-spot incorporating 96% of breakpoints. This region also contained two hotspots for chromosomal gain and tandem duplications. MYC rearrangements were not randomly distributed across the spectrum of MM with an excess being seen in hyperdiploidy (76% of rearranged samples, P & lt;0.0001). Importantly, 67% (207/308) of cases with a MYC translocation involving 5 or less chromosomes had one of the commonly known super-enhancers involved in the translocation. Gene expression analysis was used to explore the impact of these events on downstream gene expression patterns. The results showed that inter- and/or intra-chromosomal rearrangements were associated with a significantly (P & lt;0.0001) higher MYC expression (4.1-fold). In patients where rearrangements were associated with additional copies of MYC there was higher expression of MYC in comparison to cases with a translocation but lacking copy number gain (P=0.04). To identify downstream genes deregulated by MYC rearrangements we compared gene expression between those with and without a translocation, independently of hyperdiploidy. Genes that showed & gt;2-fold change in expression (P & lt;0.01) included MYC and the non-protein coding oncogene PVT1 that is located next to MYC . Genes with significantly lower levels of expression were involved in B-cell biology including CD79A and AHR, or were associated with cell proliferation, migration, adhesion, apoptosis and/or angiogenesis (FGF16, ADAMTS1, FBXL7, HRK, PDGFD, and PRKD1) . Conclusions: This study confirms the central role of MYC in the pathogenesis of clinical cases of MM, and as such defining it as a critical therapeutic target. We will be able to target MYC better if we understand how it is deregulated and in this respect we show that the MYC locus rearrangements are complex and it is a hot-spot for heterogeneous inter- as well intra-chromosomal rearrangements, including complex rearrangements involving & gt;5 chromosomes. These events lead to increased MYC expression consistent with it being a driver of disease progression, particularly in the hyperdiploid subset of MM. Disclosures Mavrommatis: Celgene Corporation: Employment. Trotter: Celgene Corporation: Equity Ownership; Celgene Institute for Translational Research Europe: Employment. Davies: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Thakurta: Celgene Corporation: Employment, Equity Ownership. Morgan: Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria.

Abstract: Integrin-β7 is part of an extensive family of glycoproteins and is overexpressed in the MF subgroups in Multiple Myeloma (MM), contributing to drug resistance and poor survival. ITGB7 promotes adhesion and proliferation of myeloma cells in bone-marrow stroma by supplementing growth factors like VEGF. Here, we investigate the possible epigenetic mechanism of ITGB7 overexpression in the MF subgroup, comprising of t(14;16) and t(14;20) myeloma. Newly diagnosed MM (NDMM) patient bone marrow aspirates underwent CD138 cell selection to enrich tumor cells to & gt;98%. Samples consisted of those with a t(14;16) (n=17), t(14;20) (n=7), t(4;14) (n=9), t(11;14) (n=10) and hyperdiploidy (n=19, separated into D1 (n=12) or D2 (n=7 subgroups). NDMM patient samples were compared to plasma cells isolated from age-matched healthy donors (n=4), to determine differential changes in epitranscripts. We performed unbiased genome-wide reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs) in CpG islands, which were validated by Infinium MethylationEPIC arrays (Illumina) in conjunction with gene expression array data (U133 Plus 2.0, Affymetrix) to determine the epitranscriptomic profile in all samples. RRBS was performed using 75 bp reads to a minimum of 20 M reads per sample. Data from MM cells at the Blueprint consortium were also used for annotation of epigenetic marks. Using the RRBS data we identified 26 hypomethylated, overexpressed genes in the MF group samples, which were not present in the other subgroups. In comparison, only 1 gene, FUT7, was hypermethylated and down-regulated in the MF cluster. Most interestingly, we identified ITGB7 amongst the hypomethylated, overexpressed genes. With an in-depth analysis we identified 4 significant (p & lt;0.05) DMRs across intragenic regions of ITGB7, within a 2.5 kb region. Mean methylation across the DMRs reduced from 58% to 22% (p & lt;0.01) in t(14;16) and 39% (p=0.02) in t(14;20) with concomitant overexpression of ITGB7 in the t(14;16), 19.5-fold increase, and the t(14;20), 23.4-fold increase, subgroups. These 4 DMRs belong to CpG islands, annotated to be the part of possible intragenic enhancer site with promoter like activity, and enriched for open chromatin structures. The DMRs also align with the hotspot of H3K4me1 marks and a putative binding site of activating transcription factors such as AP1/2 or Sp1. This suggests that the identified DMRs could be within the enhancer that regulates ITGB7 overexpression through DNA-hypomethylation. ITGB7 is a known oncogenic factor in high-risk MM, contributing to cell adhesion, migration and homing. Here we show using combined DNA methylation and expression data that ITGB7 is regulated through hypomethylation of the an enhancer region in the MF subgroup in MM. Citation Format: Samrat Roy Choudhury, Cody Ashby, Ruslana Tytarenko, Yan Wang, Purvi H. Patel, Aneta Mikulasova, Michael Bauer, Shayu Deshpande, Faith E. Davies, Gareth J. Morgan, Brian A. Walker. Intragenic DNA-hypomethylation promotes overexpression of ITGB7 in MF subgroup of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5324.