A Focused High Throughput Functional Screen Identifies Novel Targets for aggressive B-Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma is an aggressive cancer that arises from B-cells and accounts for about 30-40% of all non-Hodgkin lymphoma cases. Genetically, it can be classified as single-hit (SHL: has rearrangement in one of the three genes i.e., MYC, BCL2 or BCL6, typically MYC), double-hit (DHL: involves rearrangements in two of these genes, commonly MYC and either BCL2 or BCL6), triple-hit (THL: involving rearrangements in MYC, BCL2, and BCL6 genes) or no hit (NHL: with no rearrangements in any of these genes). Double and triple hit lymphomas are aggressive with the poor prognosis. In this study, we conducted a genome-wide functional screen in single and double hit cell lines and analyzed the data in comparison with DepMap data.

Methods: The SHL cell line Raji (MYC rearranged) and the DHL cell line SU-DHL-10 (MYC and BCL-2 rearranged) with stably integrated Cas9 were transduced (in triplicate) with a genome-wide lentiviral expression library (GeCKO v2) containing 123,411 guides representing 19,050 genes. Cells selected in puromycin were cultured and the DNA from these cells sequenced to identify genes with potential to inhibit lymphoma cell growth. Target genes unique and common to SHL (Raji) and DHL (SU-DHL-10) cells were identified and investigated for functional enrichment. Dependency Map (Cancer DepMap, https://depmap.org/portal/) database was also used to identify the functional target genes for Raji and SU-DHL-10 cells. Target genes identified in SHL (Raji) and DHL (SU-DHL-10) cells in our data and DepMap data were compared for overlap and differences.

Results: Loss of function screen identified 373, 267 and 702 genes to be associated with loss of cell viability in Raji (SHL), SU-DHL-10 (DHL) and both Raji and SU-DHL-10 cell lines, respectively. Several metabolic and signaling pathways (cGMP-PKG signaling, fatty acid degradation, pyruvate metabolism and glycolysis/gluconeogenesis) were among prominent pathways associated with 702 common hits. Interestingly, the genes identified in SHL and DHL cell lines also associated with similar metabolic pathways. Elevated expression of 390 out of 702 common hits correlated with poor overall survival(OS) in aggressive B-cell lymphoma dataset; RFC5, RFC3 RPL32 and DIS3 were among top hits. Expression of 198 out of 373 Raji (SHL)-only hits correlated with OS; MYC, MED17 and MAD2L1 were among top hits. Expression of 134 out of 267 SU-DHL-10 (DHL) only hits correlated with OS; ZNF131, DDX21 and FAM122A were among top hits. Comparing our data with DepMap data in SU-DHL-10 cells, we found 599 hits common for both datasets whereas 506 and 370 were unique for our screen and DepMap data, respectively. In Raji cells, 782 hits were common in both datasets whereas 293 and 112 were unique for our screen and DepMap data, respectively. This indicates 72% and 87% overlap in our and DepMap data in Raji and SU-DHL-10 cells, respectively. Top hits (including RFC5, RFC3 RPL32, DIS3, MYC, MED17, MAD2L1, ZNF131, DDX21 and FAM122A) are currently being evaluated in detail in our laboratory.

Conclusions: Our small functional screen provides further validation of DepMap data and identifies additional promising targets to inhibit growth of lymphoma cells. Selected hits(including RFC5, RFC3 RPL32, DIS3, MYC, MED17, MAD2L1, ZNF131, DDX21 and FAM122A) which also show correlation with survival of aggressive B-cell lymphoma cells are currently being evaluated in loss and gain of function study in our laboratory.

Munshi:AbbVie, Adaptive Bio, Amgen, Bristol Myers Squibb, Celgene, GlaxoSmithKline, Janssen, Karyopharm, Legend Bio, Novartis, Oncopep, Pfizer, Recordati, Sebia, Takeda: Consultancy; Oncopep: Current holder of stock options in a privately-held company.