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Supplementary Tables from An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML

CRISPR–Cas9-based genetic screens have successfully identified cell type–dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets.Significance:There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275

Supplementary Data from An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML

CRISPR–Cas9-based genetic screens have successfully identified cell type–dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. Significance: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities. This article is highlighted in the In This Issue feature, p. 275

Inhibition of the anti-apoptotic machinery of cancer cells is a promising therapeutic approach that has driven the development of an important class of compounds termed “BH3 mimetics”. These novel small molecules mimic BH3-only proteins by antagonizing the pro-survival function of anti-apoptotic proteins, thereby inducing apoptosis in cancer cells. To qualify as an authentic BH3 mimetic, a compound must function directly on the mitochondria of a cell of known anti-apoptotic dependence, must directly and selectively inhibit the anti-apoptotic protein with high-affinity binding, and must induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis in a BAX/BAK-dependent manner. While many BH3 mimetics have entered clinical trials, the lack of a reliable validation assay to directly test the mitochondrial activity of new BH3 mimetic candidates has resulted in many misleading reports of agents touted as BH3 mimetics despite their off-target mechanisms of action. BH3 profiling probes the activity of a compound at the mitochondrial level by measuring cytochrome c release as a surrogate marker for MOMP. We propose a comprehensive biochemical toolkit consisting of BH3 profiling in parallel with high-throughput Annexin V/Hoechst viability testing to validate BH3 mimetic candidates. We tested our toolkit on eighteen different putative BH3 mimetics using a set of standardized cell lines of known anti-apoptotic dependence. Included in this set of cell lines is an apoptosis refractory BAX/BAK DKO control line to detect compounds that function independently of the BCL-2 family. Taken together, this rapid, efficient means of testing will prove advantageous as the demand for BH3 mimetics increases, particularly in the quest to identify and develop more potent MCL-1 inhibitors for use in the clinic. We strongly urge researchers utilizing BH3 mimetics in their work to use the potent and selective compounds identified with this validation toolkit instead of those lacking such potency and selectivity.

Key Points Adding venetoclax to FluBu2 reduced-intensity conditioning transplant did not impair engraftment or induce excessive graft-versus-host disease.Monitoring measurable residual disease by ultra-sensitive duplex sequencing revealed complex clonal dynamics before and after transplant., Visual Abstract, Adding the selective BCL-2 inhibitor venetoclax to reduced-intensity conditioning chemotherapy (fludarabine and busulfan [FluBu2]) may enhance antileukemic cytotoxicity and thereby reduce the risk of posttransplant relapse. This phase 1 study investigated the recommended phase 2 dose (RP2D) of venetoclax, a BCL-2 selective inhibitor, when added to FluBu2 in adult patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and MDS/myeloproliferative neoplasms (MPN) undergoing transplant. Patients received dose-escalated venetoclax (200-400 mg daily starting day −8 for 6-7 doses) in combination with fludarabine 30 mg/m2 per day for 4 doses and busulfan 0.8 mg/kg twice daily for 8 doses on day −5 to day −2 (FluBu2). Transplant related–toxicity was evaluated from the first venetoclax dose on day −8 to day 28. Twenty-two patients were treated. At study entry, 5 patients with MDS and MDS/MPN had 5% to 10% marrow blasts, and 18 (82%) of 22 had a persistent detectable mutation. Grade 3 adverse events included mucositis, diarrhea, and liver transaminitis (n = 3 each). Neutrophil/platelet recovery and acute/chronic graft-versus-host-disease rates were similar to those of standard FluBu2. No dose-limiting toxicities were observed. The RP2D of venetoclax was 400 mg daily for 7 doses. With a median follow-up of 14.7 months (range, 8.6-24.8 months), median overall survival was not reached, and progression-free survival was 12.2 months (95% confidence interval, 6.0-not estimable). In patients with high-risk AML, MDS, and MDS/MPN, adding venetoclax to FluBu2 was feasible and safe. To further address relapse risk, assessment of maintenance therapy after venetoclax plus FluBu2 transplant is ongoing. This study was registered at clinicaltrials.gov as #NCT03613532.

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancers, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro, evaluating the physiological relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to prioritize AML-enriched dependencies in vivo, complemented by the validation in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets.Statement of significanceThere is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, representing novel therapeutic opportunities.

Relapse is the leading cause of treatment failure in acute myeloid leukemia (AML) patients. FDA approval of 8 targeted therapies in the past two years has drastically altered the landscape of AML treatment. Despite this success, the duration of clinical response is limited by the frequent development of acquired drug resistance to targeted therapy. To identify mechanism of resistance and to search for therapies that overcome resistance, we adopted broadly applicable functional approach to precision medicine called “dynamic BH3 profiling” (DBP) and coupled it with RNA-seq and targeted exome sequencing technology. DBP measures drug induced early death signaling using BH3 peptides that mimic pro-apoptotic BH3 proteins. To first validate the utility of DBP as precision medicine tool we created landscapes of pharmacologic sensitivity of 17 patient-derived xenograft (PDX) AML models to a panel of 40 clinically relevant agents, together with genomic and transcriptomic profiles. Aggregated across the panel, unsupervised clustering of drug-induced apoptotic signaling using DBP by itself could segregated PDXs according to prior treatment status (PDXs from treatment naïve patients clustered distinctly from R/R PDXs). While genomic mutations and transcriptomic signature profiles between R/R and treatment naïve PDXs did not show significantly distinct clustering patterns. Next we show that DBP could predict in vivo responses of drugs of widely varying mechanism of action, including a FLT-3 inhibitor, BCL-2 and MCL-1 inhibitors (BH3 mimetics), SMAC mimetic, and BRD4 inhibitor, in 6 AML PDX models (AUC of ROC 0.8731, p Citation Format: Shruti Bhatt, Marissa S. Piosos, Elyse A. Olesinski, Binyam G. Yilma, Jeremy A. Ryan, Thelma Mashaka, Buon Leutz, Sophia Adamia, David M. Weinstock, Jacqueline S. Garcia, Anthony Letai. Reduction in mitochondrial priming drives resistance to targeted therapy in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG14.

Background: Patients (pts) with myeloid malignancies characterized by high risk mutations or cytogenetics who undergo allogeneic hematopoietic cell transplantation (HCT) have poor outcome primarily due to risk of relapse. Strategies to limit relapse include post-transplant maintenance or novel conditioning regimens. The selective BCL-2 inhibitor venetoclax (VEN), a BH3 mimetic, promotes mitochondrial apoptosis in myeloblasts in the presence of cytotoxic stress and is associated with high response rates when combined with hypomethylating agents or low dose cytarabine in untreated elderly AML. We found that VEN could induce apoptotic priming in myeloblasts regardless of poor risk mutations such as TP53 by BH3 profiling, which measures the mitochondrial response to perturbation by a panel of BH3 domain peptides. We thus hypothesized that VEN would promote the anti-leukemic effect of conditioning chemotherapy and therefore reduce the risk of relapse without undue toxicity. Methods: The primary objective of this phase 1 study is to determine the safety of adding VEN to the RIC regimen of fludarabine and busulfex (FluBu2). This study uses a 3+3 design with dose expansion. Eligible pts included: AML in CR (adverse risk per ELN or secondary AML) or MDS (t-MDS; Int-2 or higher IPSS; TP53 mutation; or RAS pathway gene mutation) or MDS/MPN (including +8; chr 7 abnl; complex karyotype; or ASXL1 mutation) with up to 10% blasts at the time of transplant. Dose-limiting toxicity (DLT) was defined as any treatment-related death, failure to engraft (defined as absolute neutrophil count (ANC) ≥ 500/μL on 2 consecutive measurements), or any gr 4 non-heme toxicity or tumor lysis syndrome from day -8 until day +28. Conditioning chemotherapy consisted of fludarabine 30 mg/m2/d, days -5 to -2 and busulfex 0.8 mg/kg bid, days -5 to -2, followed by PBSC infusion on day 0. VEN is scheduled to maximize overlap with FluBu2. VEN dose levels (DL) are 200 mg on days -8 through -3 (DL1); 200 mg on days -8 through -2 (DL2); and 400 mg on days -8 through -2 (DL3). Flow cytometry-based BH3 profiling was performed on pre-VEN treatment bone marrow in pts with measurable residual myeloblasts. NGS using a 95-gene TruSeq panel was performed on pre-VEN treatment and day +100 marrow samples. Results: Nine pts (67% male, median age of 65 y (range 41-71)) have been treated including 3 AML (1 with -17 and 2 with mutations in TP53, RUNX1, and/or ASXL1), 5 MDS (all mutant TP53), and 1 CMML (with mutant ASXL1) in DL1-DL3. 8 donors were matched unrelated and 1 was a matched related. Donors were 44% male. No DLTs were observed at DL1-DL3; dose expansion at DL3 is ongoing. VEN-related toxicities included gr 1 diarrhea (n=3), gr 1 fatigue (n=1), and gr 1 nausea (n=2). Median time to ANC ≥ 500/μL was 16 days (range 13, 25; 1 pt did not nadir) and median donor-derived myeloid chimerism was 100% (range 97, 100) at day +28. Median time to platelet ≥ 20K/μL was 14 days (range 13, 18; 3 did not nadir). 3 developed gr 1 acute graft-versus host disease (GVHD) involving skin and gastric sites. In the 4 followed beyond day +100, 3 developed chronic GVHD (2 moderate, 1 severe) and 1 died from complications of acute GVHD of the gut (stage 3) on day +183. With a median follow-up time of 5 months (range 1.3, 7.8), 2 of 9 pts had disease progression and 1 died. Five out of 6 pts with available paired day +100 samples had no detectable mutation by NGS with >200X mean coverage at the later time point (Fig A). Pt 3 had persistent mutations in TP53 at day +100. Myeloblasts that are more primed require lower doses of [BIM] or [PUMA] peptide to induce cytochrome c release. BH3 profiling of residual myeloblasts on pre-VEN treatment marrow using pro-apoptotic BIM and PUMA peptides (Fig B-C) showed differential individual baseline priming status, which we will correlate with response. Conclusions: VEN at doses up to 400 mg can be safely added to RIC conditioning with FluBu2 resulting in a high molecular negativity rate at day +100 without impairing neutrophil engraftment. Additional correlative studies include targeted deep sequencing to better evaluate measurable residual disease for individual genes and BH3 profiling analysis of myeloblasts collected pre-VEN and post-VEN/pre-FluBu2 to evaluate for a VEN-induced increase in apoptotic priming. To further minimize disease relapse in these high risk pts, we amended the trial to include maintenance therapy with a combination of VEN and azacitidine. Figure Disclosures Garcia: Abbvie: Research Funding; Genentech: Research Funding. Ryan:Vivid Biosciences: Consultancy. Letai: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; AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Lindsley:Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding; Jazz Pharmaceuticals: Research Funding. Ho:Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Omeros Corporation: Membership on an entity's Board of Directors or advisory committees. Koreth:Cugene: Consultancy; Amgen: Consultancy; Equillium: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Galinsky:ABIM: Other: Member on specialty oncology board; AbbVie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Merus Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Steensma:Summer Road: Consultancy; Arrowhead: Equity Ownership; H3 Biosciences: Other: Research funding to institution, not investigator.; Onconova: Consultancy; Astex: Consultancy; Pfizer: Consultancy; Stemline: Consultancy; Aprea: Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. Soiffer:Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Kiadis: Other: supervisory board; Mana therapeutic: Consultancy; Cugene: Consultancy; Jazz: Consultancy. Cutler:BiolineRx: Other: DSMB; Cellect: Other: DSMB; Jazz: Consultancy; BMS: Consultancy; Genentech: Consultancy; ElsaLys: Consultancy; Kalytera: Other: DSMB; Pharmacyclics: Consultancy; Fate Therapeutics: Consultancy; Incyte: Consultancy; Kadmon: Consultancy; Omeros: Consultancy. DeAngelo:Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Novartis: Consultancy, Research Funding; Abbvie: Research Funding; Glycomimetics: Research Funding; Blueprint: Consultancy, Research Funding. OffLabel Disclosure: Venetoclax is a BH3 mimetic and selective BCL-2 inhibitor that was added to standard conditioning chemotherapy (fludarabine and busulfan) on a clinical trial.