In Vivo Genetic Screen Identifies Chromatin Remodeling Gene SET As a Collaborating Oncogenic Driver in Initiation and Maintenance of Acute Lymphoblastic Leukemia

Innovative treatment options have greatly improved outcomes for acute lymphoblastic leukemia (ALL) patients over several decades, yet relapse and refractory disease remain a significant clinical challenge. The complex genomic heterogeneity of molecular subtypes contributes to poor prognosis, high morbidity, and recurrent drug resistance in relapse and refractory ALL patients. Identifying novel genetic interactions and molecular pathways capable of initiating ALL will help to develop new diagnostic criteria and to identify actionable drug targets.

Here, we developed a novel in vivo screening method using transgenic zebrafish to unbiasedly identify collaborating oncogenic drivers that when co-expressed lead to T- and B-ALL. Our high-throughput approach allows for rapid screening of collaborating oncogenic drivers across hundreds of individual animals in a cost-effective manner. Specifically, we screened a transgene pool of 68 putative oncogenes identified from relapsed human ALL for synergies at inducing leukemia in a large-scale F₀ transgenic screen. Using our approach, complex gene networks involved in cancer initiation in human patients can be rapidly interrogated and unraveled with in vivo resolution utilizing the zebrafish model. Low concentrations of transgene pools containing candidate cDNAs driven by a tissue specific immune cell promoter were co-injected into zebrafish along with a fluorescent reporter (rag2:mCherry). Animals that developed tumors express mCherry along with only 5 to 20 additional transgenes (median 15 genes), indicative of random concatemeric integration of transgenes into the genome. Using histopathologic analysis and bulk mRNA sequencing, we identified zebrafish with mCherry-labeled thymic T cell lymphomas, T-ALL, and B-ALL ((>140 animals). Analysis of transgene expression from bulk-RNA sequencing studies allowed us to nominate causative combinations of human transgenes that drive leukemia onset in the zebrafish. Most notably, we identified several unique leukemia and lymphoma transgenic zebrafish models, representing T-ALL, B-ALL, mixed-phenotype acute leukemia (MPAL), T- and B-cell lymphoma, and NK cell leukemia. Our analysis also elucidated novel synergies between the proto-oncogene SET and either activated NOTCH1 or IL7R mutations to initiate ALL.

To verify nominated collaborating oncogenic drivers as causative at inducing cancer, we next performed microinjection validation studies with: 1) rag2:SET + rag2:intracellular notch1a + rag2:mCherry (n=7, mean latency 105±16 days) and 2) rag2:SET + rag2:mutationally-activated IL7R + rag2:mCherry (n=5, mean latency 120±25 days). Both combinations lead to robust ALL formation in zebrafish, validating the oncogenic role of these factors in leukemia initiation. Importantly, lymphoma and leukemia were not detected in animals injected with rag2:SET (n=19, >180 days followed), or rag2:intracellular notch1a (n=31, >180 days followed) or rag2:mutationally-activated IL7R (n=18; >180 days followed).

SET is a multifunctional protein involved in transcriptional regulation and histone binding. While SET has been characterized in other cancers, we found that SET collaborates with known drivers to initiate T-ALL in vivo and is also highly expressed in a vast majority of human T-ALL. SET is a potent inhibitor of H3 acetylation, suggesting that epigenetic dysregulation has a critical role in ALL oncogenic transformation. Finally, to determine whether SET is also required for human ALL maintenance, we established doxycycline inducible shRNA human ALL lines (lentiviral pINDUCER10; PMID: 21307310). We found that induced knockdown of SET inhibited cell proliferation and viability in multiple T-ALL and B-ALL cell lines while also increasing apoptotic cell death and inhibiting cell cycle progression. Taken together, our work has identified a unique role for a SET in ALL initiation and maintenance. We have generated a robust transcriptome atlas of zebrafish lymphoid malignancies, representing novel transgenic zebrafish models of several acute lymphoblastic leukemia and lymphoma molecular subtypes. In addition, our unique screening approach can be further applied towards discovery of genetic factors capable of initiating pediatric leukemias, creating a molecular “roadmap” to leukemogenesis, and identify vulnerable pathways for clinical therapies.