ALBERT

Description: Clonal evolution in response to therapy is a central feature of disease relapse. This raises a fundamental question in cancer biology: what enables the relapse clone to replace the pre-treatment clone? In other words, is the increased fitness of the relapse clone due to a lower death rate during therapy (less sensitivity to therapy) or a higher growth rate following therapy (superior ability to compete during repopulation)? We sought to address this question in chronic lymphocytic leukemia (CLL), as its relatively indolent disease kinetics enable the study of serially collected samples from the same patient over time. We recently reported the genetic characterization of 278 samples from patients enrolled in the German CLL Study Group CLL8 trial (Nature, in press). These samples were collected prior to first therapy with FC or FCR, and studied using whole-exome sequencing (WES). From this cohort, we further analyzed by WES 59 patients (FC [n = 28] or FCR [n = 31]) at time of relapse. We found that clonal evolution is the rule rather than the exception (57 / 59 CLLs), with TP53 alterations found in relapse in 15 cases. This series constitutes a unique opportunity to dissect the clonal dynamics of treated CLL. We therefore quantified clone-specific death and growth rates by targeted deep sequencing of serial peripheral blood samples, beginning at pre-treatment and ending at relapse. Given the expected minimal mutation detection sensitivity (0.1-1%) by targeted deep sequencing, we only selected samples with 〉1% CLL cells by flow cytometry. Such samples were available for 23 of 59 patients, with a median of 6 samples/patient (range 3-10). Based on the mutations identified by WES in the pre-treatment and relapse samples, we designed patient-specific multiplexed assays for targeted deep sequencing (median sequencing depth - 6561). A series of normal samples were sequenced together with patient samples to account for sequencing errors. The measurements of the CLL cell fraction in the sample, by sequencing and by flow cytometry, were highly correlated (r=0.89, p

Description: The FDA-approval of potent targeted therapies has led to great changes in the therapeutic landscape of chronic lymphocytic leukemia (CLL). As a key example, venetoclax, a first-in-class BCL-2 inhibitor, leads to response in about 80% of patients with relapsed/refractory (R/R) CLL. Disease progression on venetoclax, however, has been increasingly observed, and better biologic understanding of resistance mechanisms to this agent is needed. To systematically discover the potential mechanisms of resistance to venetoclax, we performed both genome-scale loss- (LOF) and gain-of-function (GOF) genetic modifier screens in the BCL-2-driven OCI-Ly1 lymphoma cell line using CRISPR-Cas9 sgRNA and ORF libraries, respectively. Significant hits from both screens included the BCL-2 family: the LOF screen with pro-apoptotic genes (PMAIP1, BAX, BAK1, BCL-2L11) and the GOF screen with anti-apoptotic genes (BCL2L1, BCL2L2, BCL2, MCL1). In addition, the LOF screen uncovered genes in pathways relevant to lymphoid biology (i.e, NFKBIA) and lymphoid transcription factors and modulators (IKZF5, ID3, EP300, NFIA). The GOF screen also uncovered components of the energy-stress sensor PKA/AMPK signaling pathways (ADIPOQ, PRKAR2B, PRKAA2) and regulators of mitochondrial metabolism. In parallel, we performed an integrated transcriptome, whole proteome and functional characterization of an OCI-Ly1 cell line rendered resistant to venetoclax (OCI-Ly1-R) from the parental cell line (OCI-Ly1-S). RNA-seq and spectrometry-based proteomics revealed coordinated dysregulation of transcripts and proteins in the resistant line originating from genes critical to cellular metabolism, cell cycle, B-cell biology and autophagy. Of the transcripts and proteins significantly associated with the resistant cell line, only MCL-1 overlapped with the gene hits from the genome-scale screens. Treatment of the OCI-Ly-R cells with the MCL-1 inhibitor S63845 synergized with venetoclax. Given the dysregulation of proteins critical to metabolism in both the GOF screen and in OCI-Ly1-R cells, we also evaluated the role of metabolic reprogramming in venetoclax resistance. We first assessed mitochondrial respiration by measuring the oxygen consumption rate. Compared to OCI-Ly-S cells, OCI-Ly1-R cells demonstrated markedly higher respiration levels, suggesting a state of higher oxidative phosphorylation (OXPHOS). More directly, we measured oxygen consumption following venetoclax exposure. Consistent with impairment of OXPHOS by venetoclax, we observed both an immediate decrease in oxygen consumption and an immediate burst of glycolysis following venetoclax in the OCI-Ly1-S cells, but not in the OCI-Ly1-R cells. In line with these findings, the AMPK inhibitor dorsomorphin and mitochondrial electron transport chain (mETC) inhibitors synergized with venetoclax in OCI-Ly1-S cells. Transcriptome related to ID3 (identified as one of the LOF screen targets) was characterized in isogenic ID3-knockout OCI-Ly1 lines. It revealed PRKAR2B overexpression as a key effect, suggesting a role for ID3, and perhaps of other lymphoid transcription factors in regulating metabolic reprogramming associated with resistance. Indeed, exposure of ID3 knockout lines to mETC inhibitors overcame resistance to venetoclax. To determine if there is a genetic basis for the drug resistance seen in OCI-Ly1-R cells, we compared whole-exome sequencing (WES) results of DNA isolated from the OCI-Ly1-R and OCI-Ly1-S cell lines. A clear region was amplified on chromosome 1q23, which includes MCL1 and PRKAB2 (the regulatory subunit of AMPK). Similarly, a WES-based analysis of paired CLL DNA samples isolated from 6 R/R CLL patients just prior to venetoclax initiation and at time of progression on venetoclax was performed. We did not identify any non-silent somatic single nucleotide in BCL2 or its family members at baseline or at progression, despite marked clonal shifts in all patients. We confirmed the presence of the amp(1q23) as acquired at relapse after venetoclax in 3 out of 6 patients. Our study reveals that venetoclax resistance implicates changes not only for outer mitochondrial membrane (MCL-1 expression) but also for inner membrane (oxydative metabolism). Such mitochondrial reprogramming represents a new vulnerability that can potentially be exploited through combinatorial therapy with metabolic modulators to overcome resistance. Disclosures Guieze: abbvie: Honoraria; janssen: Honoraria; gilead: Honoraria. Thompson:Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Adaptive Biotechnologies: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Davids:Merck: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; MEI Pharma: Consultancy, Research Funding; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Surface Oncology: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Brown:Sun Pharmaceutical Industries: Research Funding; Abbvie: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy; Janssen: Consultancy; Sunesis: Consultancy; Roche/Genentech: Consultancy; Verastem: Consultancy, Research Funding; Boehringer: Consultancy; Loxo: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy; Genentech: Consultancy. Wierda:AbbVie, Inc: Research Funding; Genentech: Research Funding. Letai:AstraZeneca: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Vivid Biosciences: Equity Ownership. Wu:Neon Therapeutics: Equity Ownership.

Description: Activation and proliferation of chronic lymphocytic leukemia (CLL) cells depend on signals from the lymph node (LN) tumor microenvironment (TME). Separately, the genetic makeup of CLL has been closely linked to disease aggressiveness and its capacity to evolve under the selective pressures of treatment. Here, we investigated the intersection between the TME and molecular events in CLL pathogenesis. Whole exome and RNA sequencing (RNA-seq) were performed on CD19+ cells of paired peripheral blood (PB) and LN samples and matched germline DNA from 14 patients with treatment-naïve CLL. RNA-seq was also done on unsorted LN samples containing tumor and non-tumor cells from the same patients. A median of 27 (range 11-69) somatic single nucleotide variants (sSNVs) and 3 (0-10) insertions and deletions (sIndels) were detected per exome. All but one patient had copy number alterations (CNAs), most commonly del 11q and del 13q. Cancer cell fractions (CCFs) of sSNVs, sIndels, and CNAs were inferred from variant allele frequencies then clustered over the two anatomic compartments for each patient. Genetic compartmentalization (ΔCCF 〉 0.25, false discovery rate [FDR] 〈 0.1) was observed in 7 patients (50%), of whom 6 demonstrated subclonal expansion in LN. To understand factors contributing to spatial heterogeneity, we compared the tumor transcriptome based on the presence (shifted group) or absence (stable group) of an expanded subclone in LN. Most differentially expressed genes between PB and LN were shared by all patients and reflected the activation of CLL cells in the LN TME as previously shown. However, cell cycle genes (e.g. E2F2, CDC25A) were more upregulated (log2FC 〉 0.5, FDR 〈 0.05) in LN of the shifted group, while lymphocyte activation markers (e.g. CD83, CD69) were more upregulated in LN of the stable group. We hypothesized the latter finding could indicate immune-mediated control of clonal outgrowth. We therefore evaluated the expression of an 18-gene T-cell associated inflammatory signature in unsorted LN samples. This signature was originally developed as a predictive biomarker for response to immune checkpoint blockade in multiple cancer types. Unsupervised hierarchical clustering of signature genes revealed an inflamed TME in the stable group relative to the shifted group. In summary, genetic compartmentalization is a common phenomenon in CLL. Clonal equilibrium is maintained by a T-cell inflamed TME. When immune surveillance is inactivated, subclones with a competitive advantage may expand in response to support signals provided by the TME. An immunotherapy-based clinical study using checkpoint blockade to restrict clonal evolution is currently in progress (NCT03204188). This research was supported by the Intramural Research Program of the NIH, NHLBI. Disclosures Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding. Wiestner:Merck: Research Funding; Pharmayclics: Research Funding; Acerta: Research Funding; Nurix: Research Funding.

Description: Cancer evolution poses a significant challenge to the ability of therapy to eliminate malignant cells. In chronic lymphocytic leukemia (CLL), clonal evolution was reported as a key feature of disease relapse after standard chemoimmunotherapy and after targeted therapy with the BTK inhibitor ibrutinib. To study potential selection of pre-existing sub-clones, we analyzed changes in clonal architecture at early time points in patients receiving ibrutinib therapy. We therefore performed in-depth longitudinal analysis of CLL samples collected from 59 patients treated with ibrutinib alone (n=44) or in combination with rituximab (n=15). Across this cohort, we performed whole-exome sequencing (WES) on a median of 3 samples per patient with median coverage of 105x (range 50-225x). Pretreatment samples were available for all patients, with post-treatment samples at 1 and 6 months for more than 85% of patients. Additional samples were available at 2, 3, or 12 months for 22 patients. The median age of the cohort was 65 (range 33-85) years. Thirty-one patients (53%) had del(17p) by FISH, 37 patients (63%) had IGHV unmutated CLL and 32 patients (54%) had relapsed or refractory disease. We observed a median of 56 (range 17-201) somatic silent and non-silent single nucleotide variants (SNVs) and insertions and deletions (Indels) per patient. The number of SNVs and indels did not differ between patients receiving ibrutinib alone or with rituximab (Wilcoxon, P=0.2). Thirteen patients had somatic mutations of BCR pathway genes prior to treatment, including one with CARD11-L251P, previously reported to confer resistance to ibrutinib in diffuse large B-cell lymphoma. Comparing pre-treatment and the latest samples within the first year (range 56-357 days, median 168), 26 of 59 (44%) CLLs showed significant changes in cancer cell fraction (CCF) over time, defined as a shift 〉 5% CCF of the clone with the greatest change (BH-FDR

Description: Venetoclax, the first approved BH3 mimetic targeting BCL2, demonstrates high response rate in chronic lymphocytic leukemia (CLL) but resistant cases are emerging. Aside from BCL2 mutations affecting venetoclax binding, multiple lines of mounting evidence suggest a role for non-mutational mechanisms underlying resistance to this drug. By applying both CRISPR-Cas9 knock-out and ORF overexpression screens in the lymphoma cell line OCI-Ly1, we previously reported the identification of MCL-1 overexpression and of the AMPK/PKA signaling axis in altering energy metabolism underlying venetoclax resistance (Guieze, ASH 2018). Here, we report further in-depth exploration of the impact of these findings, discovered through the analysis of lymphoid cell lines, and of specimens collected from CLL patients developing venetoclax resistance. The resistant lymphoma cell lines that we generated (OCI-Ly1 and SU-DHL4 cells) displayed increased oxidative phosphorylation (OXPHOS) compared to the parental lines, measured by Seahorse assay. We instead observed that venetoclax rapidly perturbs OXPHOS in sensitive cells. This process is dependent on mitochondrial outer membrane permeabilization, as this effect is abrogated in BAX/BAK1 double knockout (KO) cells. Targeting OXPHOS was shown to synergize with venetoclax in vitro and in vivo, as combination of venetoclax and oligomicin (an inhibitor of the ATP synthase, the complex V of the mitochondrial electron transport chain), was more effective than each drug alone in reducing tumor growth of a subcutaneous NSG xenograft model based on OCI-Ly1. Among the candidate markers driving resistance identified from the genome-wide screens, we focused on AMP pathway members (AMPK and PKA) and the ID3 transcriptional regulator, given that ID3 KO cells demonstrated similar transcriptomic changes than the resistant OCI-Ly1 cells. We found that PRKAR2B (encoding a PKA subunit), already highlighted in our ORF screen, was the top transcript overexpressed when knocking out ID3. To clarify how the dominant-negative transcription factor ID3 regulates PRKAR2B expression, we performed ATAC-seq of the ID3 OCI-Ly1 knockout (vs control) lines in order to determine differential signatures of chromatin accessibility and transcription factor engagement. We showed that ID3 repression leads to genome-wide increased accessibility associated with motifs of the lymphoid transcription factor TCF3. TCF3 has previously been shown to interact with ID3 and to be involved in the transcription of ADIPOQ, which was identified in the GOF screen. TCF3 binding sites were confirmed to be present within putative enhancer regions of PRKAR2B in a B cell context. We then investigated whether our findings could be validated in patient samples. By whole-exome sequencing of matched pretreatment and venetoclax-resistant CLL samples collected from 6 patients, we did not detect any recurrent somatic mutations associated with resistance. The resistant samples from three of 6 patients, however, harbored subclones with 1q amplification in a common region encompassing the MCL1 locus. We identified 4 additional CLL cases relapsing on venetoclax with leukemia samples collected before and after relapse. By immunohistochemical staining of 9 of 10 cases for which tissue was available, we detected increased MCL-1 expression at relapse in 6 of 9 cases (p = 0.026). We furthermore confirmed the involvement of AMPK signaling by detecting evidence of AMPK, ACC and p-ACC expression in 4 of 9 patients (all p = 0.0062). ID3 expression was decreased at matched relapse samples (p = 0.0001), supporting the presence of the resistance circuit we identified above. Taken together, our results identified the increased MCL-1 expression and PKA/AMPK activation as underlying mechanisms for venetoclax resistance. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance. Disclosures Guièze: Abbvie: Honoraria; Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Thompson:AbbVie: Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Research Funding; Pharmacyclics: Research Funding; Genentech: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Davids:AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding. Brown:AbbVie: Consultancy; Acerta Pharma: Consultancy; Loxo: Consultancy, Research Funding; BeiGene: Consultancy; Catapult Therapeutics: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Sun Pharmaceuticals: Research Funding; Janssen: Honoraria; Teva: Honoraria; Morphosys: Other: Data safety monitoring board; Invectys: Other: Data safety monitoring board; Octapharma: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Juno/Celgene: Consultancy; Dynamo Therapeutics: Consultancy; Genentech/Roche: Consultancy; Gilead: Consultancy, Research Funding. Wierda:Xencor: Research Funding; Cyclcel: Research Funding; Genentech: Research Funding; Pharmacyclics LLC: Research Funding; Gilead Sciences: Research Funding; KITE pharma: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Sunesis: Research Funding; AbbVie: Research Funding; Janssen: Research Funding; Acerta Pharma Inc: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding; Loxo Oncology Inc.: Research Funding; Juno Therapeutics: Research Funding. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding; Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Mootha:Jansen Pharmaceuticals: Other: SAB, compensation; 5am Ventures: Other: SAB, compensation; Raze Therapeutics: Other: Founder, SAB, equity. Getz:MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER; Pharmacyclics: Research Funding; IBM: Research Funding. Wu:Pharmacyclics: Research Funding; Neon Therapeutics: Other: Member, Advisory Board.