Resistant T-Cell Acute Lymphoblastic Leukemias That Emerge after In Vivo Treatment with Dexamethasone Frequently Down-Regulate Glucocorticoid Receptor Protein Expression
Authors: Wandler Anica M. , Huang Benjamin , Scacchetti Alessandro , Craig Jeff , Yan Hannah , Monsalve Gabriela , Li Qing , Dail Monique , Wong Jasmine C. , Nakitandwe Joy , Weinberg Olga , Hasserjian Robert P. , Kogan Scott C. , Sampath Deepak , Downing James R. , Yamamoto Keith , Taylor Barry S. , Aster Jon C. , Zhang Jinghui , Shannon Kevin 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, with T lineage ALL (T-ALL) accounting for approximately 20% of cases. Many T-ALL patients present with high risk clinical features, and receive aggressive multi-agent genotoxic therapies that pose a substantial risk of long-term adverse health effects. Glucocorticoids (GCs) are an integral part of current treatment strategies for T-ALL and other lymphoid cancers. Despite decades of clinical use, the molecular mechanisms underlying GC efficacy and resistance are incompletely understood. Limited initial response to GCs correlates with poor outcome, and secondary GC resistance is frequently observed in relapsed patients. Thus, identifying drugs that either enhance initial responses or prevent resistance to GCs could significantly improve the treatment of T-ALL and other lymphoid malignancies. Our laboratory performed retroviral insertional mutagenesis (RIM) in wild-type (KrasWT) and KrasG12D "knock-in" mutant mice to generate a panel of primary transplantable T-ALLs that recapitulate the genetic heterogeneity found in human cancers. We used these reagents to assess the efficacy of treatment with the GC dexamethasone (DEX) alone and in combination with the PI3 kinase (PI3K) inhibitor GDC-0941. We observed a robust and significant overall response to 15 mg/kg/day DEX in cohorts of recipient mice transplanted with 10 independent parental KrasWT (n=5) and KrasG12D (n=5) T-ALLs, which was modestly enhanced by combined treatment with 125 mg/kg/day GDC-0941. Prolonged in vivo treatment resulted in outgrowth of 65 independent relapsed T-ALLs, many of which harbor novel retroviral integrations. We verified intrinsic drug resistance in a number of these relapsed leukemias by transplanting them into secondary recipients and treating these mice with 15 mg/kg/day DEX. Intriguingly, 23 (35%) of the T-ALLs that relapsed after an initial response to treatment exhibited markedly reduced GC receptor (GR) protein expression, suggesting a novel and common mechanism for evading GC-induced cell death. Similarly, analysis of T-ALL patient samples showed that low GR expression is rare in diagnostic specimens, but enriched in relapsed/refractory cases. We have identified two likely mechanisms of GR down-regulation in relapsed mouse T-ALLs including a nonsense mutation in the gene encoding GR and a retroviral integration in a putative distal GR promoter region that greatly reduces mRNA expression. We also performed transcriptome (RNA-seq) analysis in one sensitive parental and corresponding resistant KrasWT T-ALL after short-term in vivo DEX treatment, and observed a dramatic reduction in the number of differentially expressed genes in the resistant versus parental leukemia. We recently generated transcriptomes from this panel of 10 primary RIM-induced T-ALLs with the goal of identifying a GC response signature, and also to assess the ability of resistant cells that have lost or retained GR expression to activate this program. Finally, in contrast to previous studies in this genetically accurate in vivo preclinical model (Dail et al. Nature 2014), very few relapsed T-ALLs showed reduced Notch intracellular domain (NICD) expression relative to the corresponding parental leukemia suggesting that loss of Notch1 activation is not a major mechanism contributing to DEX resistance. Taken together, these data reveal an unexpected and common putative mechanism of GC resistance in T-ALL that can inform the development of treatment strategies for relapsed/refractory patients. We are currently focusing on analyzing transcriptome data and performing whole exome sequencing in order to uncover additional mechanisms of resistance to DEX, and then using these data to identify and investigate targeted inhibitors that might overcome GC resistance in vivo.

Disclosures
Dail: Genentech, Inc.: Employment. Sampath:Genentech: Employment.