Abstract 2450: Single-cell profiling reveals programs mediating oncogenic kinase-independence and minimal residual disease in BCR-ABL-rearranged acute lymphoblastic leukemia
Authors: Murakami Mark A. , Winter Peter S. , Navia Andrew W. , Bilal Laura L. , Strouf Haley , Shigemori Kay , Gupta Alejandro , Mirza Mahnoor , Leahy Catharine S. , Calistri Nick , Jones Kristen , Scoyk Alexandra Van , Liu Huiyun , Powers Foster , Kimmerling Robert , Stevens Mark , Stevenson Kristen E. , Manalis Scott R. , Weinstock David M. , Shalek Alex K. 
Published: August 2019

Abstract
Targeted inhibitors of oncogenic kinases have transformed cancer therapy by inducing high rates of clinical response across diverse tumor types, yet they cure few patients due to the persistence of minimal residual disease (MRD), which seeds relapse. Characterizing MRD biology would promote development of more effective therapies tailored to relapse-initiating cells. However, these efforts have been complicated by the technical challenges of isolating and profiling low-frequency tumor populations. We overcome these hurdles by measuring the mass and short-term growth rate of single cells ex vivo using a series of suspended microchannel resonators (SMR) followed by single-cell RNA-seq of the same cells. This platform enables measurement of low input samples to facilitate characterization of MRD subpopulations and functionally drug test MRD-specific vulnerabilities. PDX models of BCR-ABL-rearranged acute lymphoblastic leukemia (BCR-ABL ALL) treated with targeted kinase inhibitors provide a tractable experimental system in which to deploy this platform to interrogate in vivo tumor evolution, MRD, and acquired resistance. We conducted a statistically powered 4-arm randomized phase II-like trial combining ponatinib and the novel allosteric BCR-ABL inhibitor ABL001 in a panel of 13 PDX models of BCR-ABL ALL (total evaluable mice: n=10 vehicle, n=14 ABL001, n=30 ponatinib, n=33 combination). Six of 30 animals receiving ponatinib (20% [95% CI, 8-39%]) and a significantly higher proportion receiving combination (10 of 33, 30% [95% CI, 16-49%]) met the primary endpoint of progression-free survival at 120 days on treatment (p=0.030). No mice in the vehicle or ABL001 arms met this endpoint, and all mice eventually progressed or were found to harbor MRD when sacrificed after treatment courses of up to 248 days. These durable remissions prior to relapse allowed us to delineate in vivo tumor evolution, focusing on MRD, via serial bone marrow aspirates. Drug-persistent marrow-resident leukemia cells exhibit strong enrichment for cellular quiescence programs that extend through MRD and into relapse. These programs are driven by expression of genes known to govern quiescence in hematopoietic stem cells (e.g., EGR1, NR4A1), and their expression correlates with a rare subset of biophysically small cells (mass &lt;25 pg) detected in pretreatment specimens. At MRD these small cells can be subdivided into at least two transcriptionally distinct populations, one poised to re-enter the cell cycle and likely to seed relapse, the other senescent (e.g. CDKN2A high) and therefore unlikely to contribute. Leukemia cells at relapse maintain low mass despite cell cycle re-entry. We are integrating these MRD quiescence programs with genetic and transcriptional mechanisms of tumor progression to develop rational therapies for overcoming MRD in BCR-ABL ALL.
Citation Format: Mark A. Murakami, Peter S. Winter, Andrew W. Navia, Laura L. Bilal, Haley Strouf, Kay Shigemori, Alejandro Gupta, Mahnoor Mirza, Catharine S. Leahy, Nick Calistri, Kristen Jones, Alexandra Van Scoyk, Huiyun Liu, Foster Powers, Robert Kimmerling, Mark Stevens, Kristen E. Stevenson, Scott R. Manalis, David M. Weinstock, Alex K. Shalek. Single-cell profiling reveals programs mediating oncogenic kinase-independence and minimal residual disease in BCR-ABL-rearranged acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2450.