TP53 CRISPR/Cas9 Reveals Strict BAX Dependence in the Response to BH3 Mimetic Targeting MCL1

Background

In multiple myeloma (MM), 1q gain/amplification (1q⁺) and deletion of the short arm of chromosome 17 (del17p), encompassing MCL1 and TP53, respectively, as well as the t(4;14) translocation are adverse events that reduce both event-free survival and overall survival of patients. The genome gatekeeper p53 is well known for its multiple roles in the regulation of mitochondrial apoptosis: p53 induces early direct transactivation of the BH3-only protein PUMA (BBC3) and the death effector BAX (BAX) and late direct-to-indirect transactivation of NOXA (PMAIP1) and BAK (BAK1). To investigate the direct impact of loss of p53 function in myeloma cells, we generated TP53 CRISPR/Cas9 isogenic cells from cell lines harboring t(4;14) and 1q⁺.

Materials and Methods

To knockout the expression of all p53 isoforms in two TP53^wt t(4;14)⁺ 1q⁺ myeloma cell lines, NCI-H929 and XG7 cells were sequentially transduced with 2 lentiviruses expressing Cas9 and a doxycycline-inducible TP53-specific sgRNA targeting exon 5. TP53^-/-clones (n=3) and control clones (n=3) were derived for each cell line by limiting dilution.

Results

RNAseq analysis was performed in triplicate in the 12 clones of the two MM cell lines: analysis of the 6 TP53^-/-clones versus the 6 control clones showed significant changes (log2FC>0.4, p>0.05) in the expression of 92 genes under steady-state conditions (and of more than 3000 upon nutlin3a-induced p53 accumulation) : 27% of the genes were known to be direct p53 target genes, 39% were known to be indirectly regulated, and 35% of the genes were not identified as p53 targets in solid cancer cell lines (Andrysik Z Genome Res 2017). Among the 51 downregulated genes, only 2 belonged to apoptosis pathways, namely BAX and TNFRSF10B, both direct early targets of p53. Of note, the expression of BAK1, PMAIP1 or BBC3 was not significantly modified. To directly assess the impact of BAX decrease in the 6 TP53^-/- clones, we used 3 BH3 mimetics specific for BCL2, Venetoclax, MCL1, S63845, and BCLXL, A1155463. The XG7 and NCI-H929 cell lines were predominantly resistant to Venetoclax and to A1155463, and TP53 knockout did not alter responses. In contrast, as compared to control clones, the response to S63845 was decreased 5.7 to 7.4-fold in NCI-H929 TP53^-/- (LD₅₀ median values increased from 7 to 40nM, p<0.0001) and XG7 TP53^-/- clones (LD₅₀ median values increased from 25 to 186nM, p=0.0007), respectively. This result suggested a major role of BAX in the response to S63845. Indeed, BAX silencing significantly inhibited S63845-induced cell death while BAK silencing had no protective effect, demonstrating the BAX dependence of the S63845 response. Of note, this strong dependence on p53-dependent BAX expression was not limited to XG7 or NCI-H929 : in 21 TP53^abnormal myeloma cell lines, BAX expression (GEP) was 1.7-fold lower than in 11 TP53^wt cell lines (LD₅₀ median values 34 vs. 59, p=0.0004), and sensitivity to S63845 was also significantly lower in TP53^abn MM cell lines compared to MM TP53^wt (LD₅₀ median values 75 nM vs. 12 nM, p=0.0296). The resistance of TP53^-/- cells to S63845 was overcome by the addition of Venetoclax, although the death remained lower in TP53^-/- clones compared to control clones (p=0.001). BAX and BAK activation and MCL1 co-immunoprecipitation assays further demonstrated the central role of MCL1/BAX expression and complexes in the response to S63845.

In 63 consecutive independent samples from patients with MM (23 at diagnosis, 40 at relapse), 33 1q⁺ and 30 1q^-, we show that 1q gain/amplification favored response to 25 nM S63845 alone (death median 16% in 29 1q^- vs 30% in 34 1q⁺, p=0.0132) and had a trend in combination with 300 nM Venetoclax (80% vs 89%, p=0.152). Finally, we show that 1q was sufficient to induce responses to S63845 plus Venetoclax regardless of del17p status (death median 30% in 26 1q⁺ del17p^- vs. 40% in 6 1q⁺ del17p⁺ samples). To analyze the response to the combination of S63845 and Venetoclax at the single cell level, we performed scRNASeq analysis in t(4;14)⁺ 1q⁺ del17p^+/- patients and determined the transcriptomic profile of cells surviving the combination: we show that BH3 mimetics combination heterogeneously induced death in and between patient's samples and we identified a resistance signature that is under evaluation.

All these results support the S63845 / Venetoclax combination for patients with double (1q⁺, del17p) to triple (t(4;14), 1q⁺, del17p) hit.

Chiron:Step Pharma: Research Funding. Moreau:AbbVie, Janssen, Celgene, Amgen, and Sanofi: Honoraria. Pellat-Deceunynck:Step Pharma: Research Funding.