Apoptotic rewiring via PI3K hyperactivation as a therapeutic strategy in B-cell malignancies Free

Background and Significance: PTEN is a critical negative regulator of PI3K/AKT signaling that dephosphorylates PI(3,4,5)P3 to PI(4,5)P2 and is among the most frequently deleted tumor suppressors in cancer. While PTEN-lesions occur in 5 to 18% of solid tumors and hematological malignancies, these lesions are not found in B-cell malignancies that originate from pre-germinal center stages of development, i.e. B-ALL, mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL).

Results: Unlike in other cancer types, genetic deletion and small molecule PTEN-inhibition revealed that B-ALL, MCL and CLL cells are uniquely dependent on PTEN for survival. Mechanistically, PTEN is essential in developing B-cells as it suppresses PI3K-hyperactivation that would otherwise trigger negative selection of autoreactive B-cell clones and cell death. Here, we show that loss of PTEN rewires the apoptotic network in B-cell malignancies, rendering cells dependent on the anti-apoptotic protein BCL2. This dependency creates a therapeutic vulnerability, enabling synergistic activity between the BCL2 inhibitor venetoclax and PTEN loss, and can be leveraged to overcome venetoclax resistance.

To investigate how PI3K hyperactivation affects genetic dependencies in B-cell malignancies, we performed a whole-genome CRISPR knockout screen in a B-ALL cell line following PTEN deletion. BCL2 and BCL2L1 (encoding BCL-xL) emerged as top-ranking synthetic lethal genes. To validate these findings, we treated a panel of B-ALL and MCL cell lines with the PTEN inhibitor SF1670 in combination with either the BCL2 inhibitor venetoclax or the dual BCL2/BCL-xL inhibitor navitoclax. Averaging across all tested cell lines, the highest Zero Interaction Potency (ZIP) synergy scores were 44.43 (MCL; n=5) and 34.66 (B-ALL; n=3) for venetoclax, and 35.51 (MCL; n=3) and 33.92 (B-ALL; n=3) for navitoclax combinations. All combinations exceeded the ZIP threshold of 20 for strong synergy, which measures synergy as exceeding the expected additivity of the two drugs. Notably, in MCL cell lines, the SF1670 and venetoclax combination exhibited greater synergy than the established combination of the BTK inhibitor ibrutinib and venetoclax (ZIP score: 30.62; n=4).

Consistent with the observed synergy, venetoclax and SF1670 led to a pronounced increase in cleaved PARP and caspase-3 in B-ALL and MCL cell lines. Additionally, cell death was rescued by the pan caspase inhibitor Z-VAD-FMK, confirming the caspase-dependent mechanism. Western blotting following SF1670 treatment revealed considerable reduction in MCL-1 and BCL-xL, while BCL2 levels remained stable or exhibited a modest increase, suggesting a shift towards BCL2 dependence. Dynamic BH3 profiling in B-ALL and MCL cell lines further confirmed that PTEN inhibition significantly enhanced apoptotic priming and BCL2 dependency in a dose dependent manner. These data collectively indicate that PTEN inhibition sensitizes B-cell malignancies to venetoclax by driving the anti-apoptotic dependency towards BCL2.

To genetically validate these findings, Pten^fl/fl B-ALL cells were transduced with inducible Cre, enabling conditional deletion of Pten. Following Pten deletion, cells were treated with increasing doses of venetoclax. Consistent with our pharmacologic data, Pten loss significantly enhanced venetoclax sensitivity in murine B-ALL. Additionally, in competitive growth assays, venetoclax treatment exacerbated cell death in Pten-deleted cells, reinforcing the notion that Pten-loss increases functional dependence on BCL2 for survival.

Given the enhanced BCL2 dependency induced by PTEN inhibition, we tested whether PI3K hyperactivation could resensitize venetoclax-resistant cells to BCL2 inhibition. Treatment of venetoclax-resistant MCL cell lines with SF1670 and venetoclax demonstrated strong synergy (ZIP score: 29.18; n=2), effectively restoring BCL2 sensitivity. These findings highlight the therapeutic potential of targeting PI3K/AKT hyperactivation via PTEN inhibition as a strategy to overcome acquired venetoclax resistance in B-cell malignancies. Conclusions: PI3K hyperactivation via deletion or pharmacological inhibition of PTEN rewires the apoptotic landscape of B-cell malignancies towards BCL2 dependence. Consistent with this shift, combined PTEN and BCL2 inhibition elicits strong synergy, resensitizes venetoclax-resistant cells, and defines a mechanistic basis for a highly effective therapeutic strategy.