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The proliferative burst of B lymphocytes is essential for antigen receptor repertoire diversification during the development and selective expansion of antigen-specific clones during immune responses. High proliferative activity inevitably promotes oncogenesis, the risk of which is further elevated in B lymphocytes by endogenous gene rearrangement and somatic mutations. However, B-cell–derived cancers are rare, perhaps owing to putative intrinsic tumor-suppressive mechanisms. We show that c-MYC facilitates B-cell proliferation as a protumorigenic driver and unexpectedly coengages counteracting tumor suppression through its downstream factor TFAP4. TFAP4 is mutated in human lymphoid malignancies, particularly in >10% of Burkitt lymphomas, and reduced TFAP4 expression was associated with poor survival of patients with MYC-high B-cell acute lymphoblastic leukemia. In mice, insufficient TFAP4 expression accelerated c-MYC–driven transformation of B cells. Mechanistically, c-MYC suppresses the stemness of developing B cells by inducing TFAP4 and restricting self-renewal of proliferating B cells. Thus, the pursuant transcription factor cascade functions as a tumor suppressor module that safeguards against the transformation of developing B cells.

Background: Previous studies indicate that mutations in signaling (e.g., receptor tyrosine kinases and RAS pathway members) and transcription factor genes are more common in secondary acute myeloid leukemia (sAML) than myelodysplastic syndrome (MDS), suggesting a role in disease progression. However, our understanding of the timing and order of mutation acquisition in these genes remains incomplete without analyzing paired MDS and sAML samples from the same patient. Defining the role of signaling gene mutations during progression should provide biologic insight into clonal evolution and help define prognostic markers for MDS progression. Methods: We banked paired MDS and sAML (and matched skin) samples from 44 patients (median time to progression: 306 days, range 21-3568). We sequenced 44 sAML (+ skin) samples for 285 recurrently mutated genes (RMGs) and 12 samples were selected for enhanced whole genome sequencing (eWGS, genome with deep exome coverage) of MDS and sAML samples (+ skin) to determine clonal hierarchy. Somatic mutations in these 12 samples were validated with high coverage error-corrected sequencing, and clonality was defined in MDS and sAML samples using mutation variant allele frequencies (VAFs). Additionally, error-corrected sequencing for all sAML RMG mutations, plus 40 additional genes, was performed on 43 of the MDS samples. Single cell DNA sequencing (scDNAseq, Mission Bio) was performed on 6 samples. Results: We identified 32 signaling gene mutations in 15 of the 44 sAML samples, with only 11 of 32 mutations (34%) detected in the initial, paired MDS sample (limit of detection; We next asked if low-level ( Finally, we observed that several MDS (n=6) and sAML (n=10) samples had multiple signaling gene mutations, and it was not always clear whether they occurred in the same subclone. We performed scDNAseq of 6 sAML samples with multiple signaling gene mutations (2-4/case). In 5 of 6 cases the signaling gene mutations did not occur in the same subclone. One sample contained 2 subclones with a NRAS and a PTPN11 mutation, with a separate subclone harboring an additional NRAS mutation. In sum, the co-occurrence of two signaling gene mutations in the same subclone is rare, indicating that the presence of multiple signaling gene mutations may be functionally redundant or detrimental to leukemia cells. Conclusions: Rare cells containing signaling gene mutations are present in nearly half of MDS patients who progress to sAML. The high frequency of signaling gene mutations and diverse patterns of clonal evolution (including the loss of one mutation and acquisition of another), suggest that signaling genes are a major driver of progression to sAML. The paucity of subclones with multiple signaling gene mutations suggests a therapeutic vulnerability for mutant cells. Disclosures DiPersio: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Jacoby:AbbVie: Research Funding; Jazz Pharmaceuticals: Research Funding.

Background: Expansion of one or more subclone occurs during progression from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (sAML). Existing data suggest that acquired mutations in myeloid transcription factor (e.g., RUNX1, CEBPA, WT1) and signaling genes (e.g., receptor tyrosine kinases or RAS pathway genes) contribute to clonal evolution and the rising blast count that defines progression to sAML. While signaling gene (SG) mutations are typically acquired later in disease progression, our understanding of when transcription factor (TF) mutations occur, in what clone they occur (e.g. founding clone or subclone), and whether TF-mutated clones undergo further clonal evolution remains incomplete. This is largely due to the limited number of paired MDS and sAML samples analyzed, the limitation of current sequencing technology and the lack of serial samples, and incomplete characterization of tumor clonality. Methods: We banked paired MDS and sAML (plus skin) samples from 44 patients who progressed from MDS to sAML (median time to progression 306 days, range 21-3568). We sequenced sAML and skin samples for 285 recurrently mutated genes (RMGs) and genotyped the paired MDS sample in patients with TF and/or SG mutations. Twelve patients were selected for enhanced whole genome sequencing (eWGS) of MDS and sAML samples (plus skin) to characterize tumor clonality. Somatic mutations were validated using error-corrected sequencing and clones were identified in MDS and sAML samples using mutation variant allele frequencies (VAFs). We tracked clonal evolution by sequencing serial samples between MDS and sAML. Results: The frequency of both TF and SG mutations were elevated in the 44 sAML patients compared to our previously sequenced cohort of 150 independent de novo MDS patients (signaling: 36% vs. 15%, transcription factor: 30% vs. 11%, respectively, p We next addressed whether TF mutations were present in a founding clone or subclone at MDS. As the accuracy of discriminating founding clones and subclones increases with the number of detected mutations, we performed eWGS on 12 pairs of MDS and sAML samples to determine if TF and SG mutations were subclonal and how subclones evolved during progression. We validated a median of 596 (range: 305-1382) mutations per MDS sample and 582 (range 305-1373) mutations per sample at sAML. Mutation VAFs were used to cluster mutations and identify clones. A median of 4 clones (range: 2-6) were detected at MDS compared to 5 (range: 2-8) at sAML, with no patient showing a decrease in the number of clones at progression. When combined with cytogenetic/copy number alterations, 11/12 cases showed the expansion of a subclone during progression. Nine SG and 8 TF mutations (n=17 total) were detected in the 12 sAML samples. In total, 16/17 (94%) TF or SG mutations occurred in subclones, including 14 in subclones that expanded during progression to sAML. Only 1 TF mutation occurred in a founding clone, and 3/4 TF-mutated subclones gave rise to a new subclone, 2 containing a new RMG. Conclusions: Data from 44 patients shows that nearly half of sAML patients have transcription factor and/or signaling gene mutations. While both are enriched at sAML compared to MDS, TF and SG mutations show different timing of mutation acquisition. TF mutations are often present at MDS in subclones indicating that they may serve as a biomarker at MDS for later progression. In comparison, SG mutations are only rarely detected at MDS and are acquired between MDS and sAML. Additionally, for cells containing both TF and SG mutations, the TF mutation is typically acquired first. Despite these differences in acquisition, both TF and SG mutations occur in subclones that often expand and continue to clonally evolve during disease progression, suggesting that they may contribute to the rise in blast count. Disclosures No relevant conflicts of interest to declare.

Background: MDS is composed of a dominant founding clone and typically one or more subclones that descend from the founding clone. Using whole genome sequencing (WGS) of secondary AML patients who were initially diagnosed with MDS, we previously showed that AML progression always coincided with expansion of at least one subclone. However, this emergent subclone was not always detectable in the antecedent MDS samples using high-coverage sequencing. It is currently unclear whether these emerging secondary AML-specific clones represent new mutations acquired in cells during treatment or selection of a rare subclone that was below the level of detection in the diagnostic MDS sample. Identification of such pre-existing subclones at MDS diagnosis could provide important prognostic information. Using an ultra-sensitive, error-corrected sequencing approach coupled with high coverage depths, we sought to determine whether secondary AML-specific mutations could be detected in antecedent MDS samples. Methods: We studied 4 MDS patients who eventually progressed to secondary AML (mean 601 days to transformation; range 131-955) that had DNA from initial MDS banking, secondary AML, and skin (as a normal control). DNA from the secondary AML sample was whole genome (3 cases) or exome sequenced (1 case). Custom enrichment panels were designed to target somatic mutations identified in the secondary AML samples and used to sequence the prior MDS samples. All 4 MDS cases had at least one subclone present in the secondary AML sample that was not detected by standard panel-based re-sequencing of the initial MDS sample (539x average coverage). We designed custom molecular barcode-based reagents to perform high-sensitivity, error-corrected sequencing targeting mutations that were detected only at progression to secondary AML. These mutation sites were then sequenced to ultra-deep coverage in prior MDS samples. To determine the sensitivity and specificity of the assay, we first sequenced known dilutions of cell line DNA and showed a sensitivity of 75% to detect variant allele fractions (VAFs) of 0.06% (one mutant cell in 800 cells) while maintaining a specificity of >99.9%. Only reads with unique molecular barcodes that were seen at least three times and had >90% identical reads within a read family were considered. Samples were sequenced to an average total coverage depth of 187,000x (range 107k-474k fold) corresponding to 10,398 unique molecular barcodes of which 3,549 passed filtering (at least two unique read families were required to call a variant), resulting in a mean predicted maximum VAF sensitivity of 0.05% (one mutant cell in 1,000 cells). Results: Using error-corrected barcode sequencing all 4 MDS cases had a subset of mutations detected that were originally only detected in the secondary AML; the mean VAF of detected mutations was 0.25% (range 0.02% to 3.9%) in the antecedent MDS samples, corresponding to 1 mutant cell in 200 cells. However, not all of the 'secondary AML-specific' mutations were detected in MDS samples. On average, only 17% (range 10-39%) of the 'secondary AML-specific' subclone mutations were detected in each MDS sample. This suggests that while some mutations pre-exist in an ancestral subclone in the MDS sample, others are acquired during the intervening months to years between MDS and progression to secondary AML. As an example, in two patients who were given decitabine prior to secondary AML progression, mutations detected in the secondary AML subclone that were not detected in the antecedent MDS sample using error-corrected barcode sequencing were more frequently C->G transversions compared to commonly acquired C->T transitions seen in MDS samples (p=.005; Mantel-Haenszel test). Increased C->G transversions are associated with DNA damage induced by decitabine, suggesting that in these two patients an ancestral subclone pre-existed in MDS that acquired additional decitabine-induced mutations during treatment (i.e., C->G transversions). Conclusion: Collectively, the data indicate that very rare subclones pre-exist at MDS diagnosis. These pre-existing subclones can subsequently give rise to the dominant clone at progression to secondary AML. Additional mutations -- some induced by chemotherapy treatment --- are also acquired in ancestral subclones that pre-exist in MDS samples. The combination of subclonal mutations may ultimately contribute to disease progression. Disclosures Jacoby: Sunesis: Research Funding; Quintiles: Consultancy; Celgene: Speakers Bureau.

Background: MDS is a genetically complex, oligoclonal disease consisting of a founding clone and typically one or more subclones derived from the founding clone. Previously we have shown that in MDS patients treated with chemotherapy, a minor subclone present at diagnosis can expand during disease progression from MDS to secondary AML, highlighting the clinical implications of clonal heterogeneity. Whether a similar pattern of clonal evolution occurs in MDS patients that relapse following allogeneic hematopoietic stem cell transplant (alloHSCT) is not known. Methods: We identified 9 MDS patients who progressed after receiving either a myeloablative (n=3) or reduced-intensity (n=6) alloHSCT (median time to progression 309 days, range 98-881). We performed enhanced exome sequencing (EES) to define the clonal architecture of 23 tumor samples at the following clinical landmarks (with matched skin as a source of normal DNA): diagnosis, Results: Averaged sequencing coverage depth was 246x for tumors subjected to EES; 537x for validation sequencing, and 24,150x total and 5,180x unique for the ultra-deep sequencing. In all cases, we observed that mutations found in the diagnostic founding clone were always detected at relapse. However, using SNVs, INDELs, and copy number analysis, we show that the dominant clone at relapse was often derived from a population that was subclonal at presentation. We observed the following, non-mutually exclusive patterns of clonal evolution at relapse: i) A subclone expanded or emerged and became the dominant clone at relapse as compared to presentation (n=6). In 2 of these cases, the subclone contained mutations that were not detected at presentation even via ultra-deep sequencing. ii) A subclone was cleared with alloHSCT (defined as VAF Finally, we used ultra-deep sequencing to determine if mutations (i.e., tumor cells) could be detected at day 30 post-alloHSCT in 7 of the 8 patients with no evidence of disease, who had available data. Mutations were detected in 6 of 7 patients. The average detectable mutation VAF per patient was 0.37% (ranged from 0.04% to 0.93%)(i.e., 1 mutant cell in 135). Conclusion: Complex clonal dynamics and clonal evolution are observed at relapse post-alloHSCT for MDS. Although minor subclones rise and may become the dominant clone at relapse, mutations present in the dominant (i.e., founding) clone of the diagnostic MDS sample pre-alloHSCT are always detected at relapse. This is similar to the pattern of clonal evolution previously observed for MDS progression to secondary AML following chemotherapy. These observations have implications for targeted therapy and tumor burden monitoring. Ultra-deep sequencing can detect persistent or emerging mutations at early time-points post-alloHSCT that are associated with subsequent relapse. The predictive value of detecting persistent mutations early after post-alloHSCT merits testing in future studies. Disclosures Jacoby: Quintiles: Consultancy; Sunesis: Research Funding; Celgene: Speakers Bureau. DiPersio:Incyte Corporation: Research Funding.

Hematopoietic cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) contain gene mutations that are variably distributed between the founding clone and daughter subclone(s). Traditional response criteria in MDS and AML are based on bone marrow morphology and may not accurately reflect antitumor activity and clinical benefit in patients treated with hypomethylating agents. We used digital sequencing of serial bone marrow samples to monitor tumor burden and to characterize the changes in the clonal structure of MDS and AML that occur during treatment with epigenetic therapy. We hypothesized that digital sequencing may provide an alternative measure of antitumor activity and identify the persistence or emergence of resistant clones during treatment which mediate disease relapse. We conducted a phase I/II study in older adults (age ≥ 60) with advanced MDS (IPSS ≥ 1.5) or AML. Subjects received a combination of decitabine 20 mg/m2 on d1-5 with the histone deacetylase inhibitor, panobinostat 10-40 mg po 3x/week every 28 days for up to 12 cycles. Serial bone marrow samples were collected for digital sequencing at baseline, after every 2 cycles of treatment and at the time of relapse. A total of 52 patients, 14 with MDS and 38 with AML were enrolled in this study. For AML patients, 10% achieved a complete remission (CR+CRi) with an additional 18% of patients achieving a morphologic leukemia-free state (mLFS) using IWG response criteria. For patients with MDS, 14% achieved a CR and 21% achieved a marrow CR. We identified 9 MDS and 16 AML patients that had banked, paired bone marrow and skin (as a source of normal DNA) samples and a somatic mutation in at least 1 of 54 recurrently mutated MDS/ AML genes. DNA was enriched for 285 genes commonly mutated in MDS and AML (n=24 patients) or whole exome probes spiked-in with the 285 genes (enhanced exome sequencing; EES) (n=7 patients), and sequenced on a HiSeq2000 instrument with 2x101bp reads. We detected an average of 4.9 SNVs and indels per patient (range 1-15) when only the 285 gene panel was used, compared to 27.4 mutations per patient (range 9-43) using EES. Ten genes were mutated in at least 3 pre-study samples. The presence of a TP53 mutation (N=8) was associated with a trend towards achieving a response (p=0.09). We then analyzed variant allele frequencies (VAF) of mutations in serial samples. We observed five distinct patterns that were associated with different clinical responses, including i) AML patients achieving a CR+CRi (n=2): mutation VAFs were undetectable by cycle 2 using standard sequencing, ii) AML with mLFS (n=2): mutation VAFs remained detectable but decreased to 10%, and v) AML with treatment failure (n=5): mutation VAFs were essentially unchanged and remained >30%. We observed responding patients can have persistent measurable clonal hematopoiesis for at least one year without disease progression. Sequencing also revealed selective AML subclone clearance in a patient with treatment failure, nominating a set of mutations that may mark super-responder clones. We observed that the blast percentage decreases prior to mutation VAFs in some patients, suggesting that the differentiation of blasts could falsely underestimate tumor burden. Finally, sequencing revealed that tumor burden can be measured even in patients achieving a CR. Using an ultra-sensitive barcode sequencing approach, we sequenced 1 MDS and 1 AML patient achieving a clinical and molecular CR (based on standard sequencing). We detected extremely rare TP53 mutations months to years prior to disease relapse (VAFs = 0.23% in MDS and 0.05% in AML during a CR - equivalent to a sensitivity of 1 in 2000 heterozygous mutant cells). While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone drives relapse or progression from MDS to secondary AML. Digital sequencing provides an alternative measure of disease response which may augment traditional clinical response criteria and should be explored in future clinical trials. Disclosures Uy: Novartis: Research Funding. Off Label Use: Panobinostat in MDS/AML. Duncavage:Cofactor Genomics: Consultancy; DI&P Consulting: Consultancy. Jacoby:Sunesis: Research Funding; Novo Nordisk: Consultancy. Abboud:Teva Phamaceutical: Research Funding.

Recent whole genome sequencing efforts by our group and others have identified recurrent mutations in the DNA methyltransferase DNMT3A in various hematologic malignancies, including acute myeloid leukemia (AML), MPN, MDS, CMML, and T-ALL. The most common point mutation in AML, R882H, is nearly always heterozygous in AML, and has been demonstrated to have a dominant negative effect on the methylation activity of the remaining wild-type allele (Russler-Germain et al. Cancer Cell 2014). However, other heterozygous mutations produce frameshifts, premature stop codons, or deletions of the entire coding sequence of the gene, strongly suggesting that these mutations lead to simple haploinsufficiency for DNMT3A. In order to test the hypothesis that Dnmt3a haploinsuffiency may initiate AML, we performed a long-term tumor watch comparing wild-type mice (Dnmt3a+/+) to mice carrying one wild-type Dnmt3a allele and one knockout allele that contains a neomycin-resistance cassette inserted into the sequence coding for the catalytic domain of the protein, producing a true null allele (Dnmt3a+/- mice). At 6 weeks of age, Dnmt3a+/- mice have normal hematopoiesis, with no detectable differences from wild-type littermates in myeloid, lymphoid, erythroid, or stem/progenitor populations in the bone marrow or spleen. However, after 1.5 years of age, 15/43 Dnmt3a+/- mice (35%) became moribund and were euthanized for pathologic evaluation (Figure 1). Affected mice generally exhibited massive splenomegaly with myeloid infiltrates in spleen, liver, and other extramedullary organs. At conclusion of the tumor watch at two years, all remaining mice were euthanized, and similar pathologic findings were observed in an additional 9 Dnmt3a+/- mice, for an overall disease penetrance of 24/43 (56%). Flow cytometric evaluation of splenic tumors demonstrated positivity for the myeloid markers Gr-1 and CD11b, and the presence of a subpopulation of cells double-positive for the mature myeloid marker Gr-1 and the progenitor cell marker CD34. Based on flow cytometric and morphologic findings, 16 splenic tumors were definitively classified according to the Bethesda criteria and were found to exhibit myeloid proliferation/MPD (11/16), myeloid leukemia with maturation (2/16), MPD-like myeloid leukemia (2/16), or myeloid sarcoma (1/16). 6 of 16 tumors tested were able to successfully engraft and lead to lethal disease in sublethally irradiated wild-type recipients, providing further evidence that these tumors represent transplantable, cell-autonomous myeloid malignancies. Analysis of exome sequencing data from 5 primary tumor samples is ongoing. One MPD-like myeloid leukemia was engrafted into four separate wild-type recipients, and all 5 tumors were sequenced. Collectively, this set of samples was found to contain mutations in the Ras/MAPK pathway, including the canonical gain-of-function mutation Kras G12C, a Ptpn11 E76K mutation, and a missense mutation in the tumor suppressor Neurofibromatosis 1 (Nf1). A second, independent primary tumor was also found to contain an activating Kras G13D mutation. Examination of the Dnmt3a locus in all 5 sequenced samples revealed no evidence of mutations or loss of heterozygosity of the residual wild-type Dnmt3a allele. Importantly, 9/51 AML samples with DNMT3A mutations in the TCGA AML cohort also contained activating NRAS or KRAS mutations. These data strongly suggest that Ras/MAPK pathway mutations can cooperate with Dnmt3a haploinsufficiency to induce AML in C57Bl/6 mice and in humans. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.