Systematic Functional Characterization for Loss of Function Events Cooperating with TP53 Loss in High-Risk 17p13(del) Multiple Myeloma

Del(17p), a high-risk subset of multiple myeloma (MM), remains, despite therapeutic advancements, a major clinical challenge, especially with biallelic loss-of-function lesions of TP53, the important tumor suppressor gene (TSG) on chr17p. Interestingly, del(17p) is predominantly monoallelic and typically involves a large part of or the entire p-arm. TP53 is, thus, often co-deleted with several other genes whose biological consequences remain underexplored. This study aims to dissect the biology of hemizygous del(17p) MM; and identify other 17p-resident TSGs that may cooperate with TP53 or, conversely, genes which exhibit synthetic lethality with TP53 loss.

We applied systematic in silico approach for the selection of candidate genes of interest. We analysed publicly available datasets to identify the commonly deleted region in MM patients, utilised the DepMap portal to rule out essential genes and performed survival analyses and copy number-expression level association study. These approaches with literature search led us to final 2 candidates. We performed CRISPR-based loss-of-function with gain-of-function overexpression studies to elucidate the functional role of the candidate genes in its cell autonomous state and in the context of standard-of-care therapeutics.

CoMMpass patients with double-hit TP53 lesions had the shortest overall survival (Mut/- or -/- or Mut/Mut), but those with hemizygous del(17p) without mutations on the remaining TP53 allele (WT/-) also displayed inferior clinical outcome vs cases with intact copies of chr17p, independently on the TP53 mutation status (WT/WT and WT/Mut), underscoring the importance of the remaining WT allele. Analysis of both MMRC and CoMMpass datasets revealed a total of 54 genes in the commonly deleted region. Comparison of events having del(17p) vs without, revealed that at the genome-wide level, 98% of the top 100 differentially downregulated genes were residents of chr17p, with 17% residing at the minimally deleted region. Two of the top hits were SAT2 and ZBTB4, which we then nominated for further molecular and functional characterization. Both genes exhibit recurrent DNA copy number losses in MM, that significantly correlated with their mRNA expression level, compared with other tumor types on CCLE, suggesting a putative preferential loss in MM. Transcript levels for both SAT2 and ZBTB4 were closely associated with patients’ survival in CoMMpass and various other independent datasets. Importantly, combination of low expression of TP53, SAT2, and ZBTB4 correlated with highly significant lower survival rate. Individual depletion of SAT2 and ZBTB4 via CRISPR-KO conferred growth advantage to the cells whereas overexpression abrogated the growth suppressive phenotype and enhanced apoptotic potential. At the therapeutic front, bortezomib washout assay revealed a faster growth recovery of SAT2-KO and ZBTB4-KO cells at the post washout period compared to control cells. Longitudinal exposure of MM cells with lenalidomide was unable to abate the growth advantage of cells with loss of SAT2 and ZBTB4, underscoring their potential role of these genes in treatment resistance.

The inferior outcome conferred by hemizygous loss of chr17p suggests that inactivation of both TP53 alleles may not be sufficient to recapitulate the full biological effect of monoallelic 17p(del) and that concomitant loss of other putative TSGs in 17p may play a role. In our study, SAT2 and ZBTB4 demonstrated potential roles in regulating growth and responses to therapeutics in MM. Our study sheds lights on the greatly underappreciated biology of high-risk del(17p) MM. Ongoing studies involve the basis for potential cooperating function of TP53 loss with SAT2/ZBTB4 and the development of therapeutic interventions to counteract this cooperation.

Chng:Hummingbird: Research Funding; Takeda: Honoraria; Novartis: Honoraria; Abbvie: Honoraria; BMS: Honoraria; Celgene: Honoraria, Research Funding; J&J: Honoraria, Research Funding; Amgen: Honoraria. Mitsiades:FIMECS: Consultancy; Secure Bio: Consultancy; Oncopeptides: Consultancy; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Abbvie: Research Funding; Arch Oncology: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Nurix: Research Funding; BMS: Research Funding; H3 Biomedicine: Research Funding; Springworks: Research Funding; Novartis: Research Funding; Fate Therapeutics: Consultancy; Ionis Pharmaceuticals: Consultancy.