Targeting Bclxl Mitigates Mcl1 Chemoresistance in Multiple Myeloma

Aim:

Despite the adoption of novel therapeutic modalities, Multiple Myeloma (MM) remains incurable. The Bcl2 inhibitor Venetoclax is active in several haematologic malignancies, but the benefits in MM patients are limited to those with the t(11;14) and/or high Bcl2 expression. These results underscore the significance of Bcl2 alternative anti-apoptotic proteins (Mcl1 and BclxL) for the survival of myeloma cells.

Method:

We validated the anti MM effect of the Mcl1 inhibitor S63845 both in vitro utilising 11 human myeloma cell lines (HMCL) and ex vivo against n=30 primary MM tumours. Comparative analysis of RNAseq between S63845 sensitive and resistant HMCL was undertaken to identify candidate proteins that potentially modulate resistance to S63845. Treatment with S63845 and rationally selected combination partners was further evaluated in vitro, ex vivo and in vivo with flow cytometry, immunoblotting and live imaging mitochondria fitness monitoring.

Results:

RNAseq identified BclxL as potential mediator of resistance to S63845 in HMCL. Immunoblotting confirmed high BclxL expression and high BclxL/BclS in S63845 resistant HMCL. Five S63845 resistant HMCL (U266, ANBL6, KMS28PE, EJM, MM1R) and primary tumours were treated with S63845 combined with the BclxL inhibitor A1331852 . Combined treatment of the HMCL demonstrated a high Bliss synergy score for all the HMCL tested (54, 42, 24, 47, 45 for U266, EJM, KMS28PE, MM1R and ANBL6 respectively) and induced synergistic killing of 80% of the primary tumours treated. Dual inhibition in U266 induced an 80% drop in intracellular ATP at 4h with an increase in active Caspases 9 and 8 (4.5 and 5 fold, respectively). Similarly, the combination induced a 78% drop in mitochondrial transmembrane potential (TMRE intensity) by 4h with live imaging revealing striking mitochondrial damage as early as 40 minutes after exposure (figure). These changes were associated with a reduction of both Mcl1 and, BclxL proteins and Bim and Bid protein levels. No changes were seen in the level of Bcl2, Bak or Bax protein expression. The combination of S63845 and A1331852 in healthy NSG mice at 12.5mg/kg proved lethal due to hepatotoxicity, arguing against the clinical utility of such an approach. However, this observed anti-MM synergistic activity was recapitulated when S63845 was combined with the already approved anti-MM therapeutic panobinostat, with the induction of a significant reduction in both BclxL and Myc protein levels at 24h, and synergistic killing of 56% of primary tumours.

Conclusion:

High BclxL expression and BclxL/BclxS ratio correlates with resistance to the Mcl1 inhibitor S63845. A combinatorial approach targeting Mcl1 and BclxL induced immediate and significant anti-MM effect both in vitro and ex vivo but proved to be toxic in vivo. Combination of the anti-MM therapeutic panobinostat in combination with S635845 recapitulated the anti-MM activity seen with A1331852 and warrants further evaluation.

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