Functional Analysis of the CML Blast Crisis Transcriptome and Epigenome Using Crispr-CAS9 and Pharmacologic Approaches

Abstract

Current models of CML blast crisis (BC) propose that expression of BCR-ABL results in genomic instability and the acquisition of genetic alterations that affect cell proliferation and survival, self-renewal and differentiation. To characterize the molecular events that underlie progression, we performed whole genome sequencing of paired samples of the same patient at CP and at BC (n = 12), as well as expression and methylation arrays of these samples and a larger validation cohort of unpaired CD34-selected samples (n = 38). Contrary to expectations, we found that the CML BC genome is relatively quiescent with regards to SNVs, indels and structural variations. In contrast, we observed widespread hyper-methylation in BC that was associated with distinct changes in expression and was independent of lineage/differentiation state. These findings suggest that in addition to genetic alterations, epigenomic events are likely to contribute substantively to BC progression.To understand the functional effects of the dysregulated transcriptome and epigenome in BC CML, we employed both pharmacologic and genetic methods to target candidate genes of interest identified in our earlier studies.To induce de-methylation of the BC genome, we treated primary samples with low doses of decitabine, a DNMT inhibitor. We found that decitabine impaired colony formation ability of BC CD34+ progenitors and concomitantly activated regulators of myeloid differentiation that were both hyper-methylated and down-regulated in BC CD34+ progenitors, such as MPO and KLF1. These results suggest that hyper-methylation does contribute to BC CD34+ progenitor function, and support the use of epigenetic therapies as a rational approach to targeting BC.The genetic approach we chose was a CRISPR-based in vitro pooled screen. We created a custom library targeting 200 genes, with an average of 5 sgRNAs per gene, and 50 non-targeting controls. We transduced K562 with the library and harvested samples at different time-points post-transduction/selection - Day 0, 7 and 21 - for deep sequencing. As expected, sgRNAs targeting essential genes such as MYC and MCM 2-7 were recurrently depleted in the population over time. More importantly, enriched sgRNAs targeted genes including TET2, which has been previously reported to be inactivated in myeloid malignancies, as well as novel candidates including RREB1, a transcription factor that binds to RAS-responsive elements (RREs) and may be involved in MAPK signaling. We will validate these targets by knocking them out individually and assessing their effect on the ability of CP cells to serially replate and/or engraft immune-deficient mice. DisclosuresChuah:Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Chiltern International: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Astellas: Speakers Bureau. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.