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3940 MYCN Is Required in Immature T-ALL and Bears Epigenetic Marks Associated with Cell Identity

Program: Oral and Poster Abstracts
Session: 603. Lymphoid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Lymphoid Leukemias, ALL, Diseases, Lymphoid Malignancies
Monday, December 12, 2022, 6:00 PM-8:00 PM

Gabriela Cristina Segat, MSc1*, Ann Chong Sun1*, Alice C Zhu2*, Marissa Foo1*, Claudia V Barreto Rodriguez1*, Rachel Wong1*, Samuel Gusscott, PhD1*, Martin Hirst, PhD2* and Andrew P. Weng, MD3

1Terry Fox Laboratory, BC Cancer, Vancouver, BC, Canada
2Canada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, Canada
3Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of pediatric and 25% of adult ALL cases and is associated with higher rate of relapse as compared to B-ALL. Although prognosis in T-ALL is excellent overall, outcomes for patients with intrinsic chemoresistance or those who relapse remains poor. Recent genome-wide studies have shed light into previously unknown oncogenes and signaling pathways; however, how different combinations of oncogenes cooperate remains unclear.

In hopes of generating more relevant experimental models, we developed an efficient and reproducible approach to generating T-ALL from primary human CD34+ cord blood (CB) cells by lentiviral transduction followed by culture first in vitro on OP9-DL1 feeders then injection into immunodeficient NSG recipient mice. This yielded aggressive disease within 12-20 weeks which was indistinguishable from spontaneously occurring human T-ALL as judged by whole transcriptomic analysis (Kusakabe et al., Nature Communications 2019). Unlike conventional mouse models of T-ALL, activated NOTCH1 alone was inefficient at transforming human cells whereas addition of LMO2, TAL1, and BMI1 (for a total of 4 delivered oncogenes, N+L+T+B) yielded high penetrance, short latency disease in vivo and showed marked growth advantage over NOTCH1-only transduced controls in vitro.

We now report whole transcriptomic analysis by RNA-seq of a collection of 75 synthetic CB leukemias generated with various combinations of N, L, T, and/or B oncogenes which yielded two major types of leukemia. One type resembled CD4- CD8- T cells, lacked TCRα/β rearrangements, and was significantly enriched for hematopoietic stem cell, inflammatory response, and MYC targets signatures, whereas the other corresponded to CD4+ CD8+ T cells, were TCRβ rearranged, and showed enrichment for Wnt/beta-catenin signalling and double positive thymocyte signatures. Clustering using a 2,824-gene list emphasizing normal T-cell development (Wong et al., Leukemia Research 2021) further characterized these two types as corresponding to earlier vs. later stages of early T-cell development. Since all synthetic leukemias carried activated NOTCH1 which drives MYC expression in T-ALL, it was not surprising that MYC transcripts were expressed consistently in all samples; however, we observed a striking elevation of MYCN expression specifically in early cluster samples as compared to late cluster samples (log2FC=3.7, padj<0.0001). Interestingly, ChIP-seq analysis of NLTB-transduced CB cells revealed enrichment for H3K4me3 marks broadly across the MYCN gene body, suggesting consistent expression across cells and thus providing a potentially critical function.

To assess for potential oncogene “addiction” to MYCN, select cell lines and primary synthetic leukemias were transduced with shRNAs or sgRNAs to knock-down (KD) or knock-out (KO) MYCN, respectively. By both approaches, bulk cell growth and/or clonogenic activity were reduced in KD/KO cells as compared to controls, with effects more pronounced in MYCN-high cells, supporting their functional dependence on MYCN. Furthermore, enforced expression of MYCN in combination with activated NOTCH1 in CD34+ CB cells resulted in increased clonogenic activity and differentiation delay in vitro but was insufficient to generate leukemia in vivo. Analysis of publicly available RNA- and ChIP-seq datasets revealed MYCN to be developmentally regulated in early T cells with highest expression in early T-cell progenitors (ETPs) and T-ALLs of less mature phenotype. Interestingly, T-ALL patients with high MYCN expression exhibited significantly shorter survival as compared to those with low MYCN (hazard ratio = 5.4, p=0.029), and was independent of designation as ETP-ALL in multivariate analyses (NCI TARGET dataset, n=265).

In summary, genetic modeling of T-ALL using primary human CB cells has revealed MYCN as a critical oncogenic effector associated with less mature phenotype tumors (e.g. ETP-ALL) and poor survival. Mechanistically, we identified broad H3K4me3 marks over the MYCN gene body suggesting that epigenetic regulation may be a key element in maintaining consistent MYCN expression across cells and defining their malignant identity. These results emphasize developmental stage-specific oncogene dependencies in T-ALL and could help motivate development of alternate therapeutic modalities.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH