Selective targeting of BCL2 with the BH3-mimetic venetoclax is proving transformative for patients with various leukemias. TP53 controls apoptosis upstream from where BCL2 and its pro-survival relatives, such as MCL1, act. Therefore, targeting these pro-survival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL2 has produced clinically relevant responses in blood cancers with aberrant TP53. However, we show that TP53 mutated or deficient myeloid and lymphoid leukemias outcompete isogenic controls with intact TP53, unless sufficient concentrations of BH3-mimetics targeting BCL2 or MCL1 are applied. Strikingly, tumor cells with TP53 dysfunction escape and thrive over time if inhibition of BCL2 or MCL1 is sub-lethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study reveals the key role of TP53 in shaping long-term responses to BH3-mimetic drugs and reconciles the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia (CLL) or acute myeloid leukemia (AML). In contrast to BH3-mimetics targeting just BCL2 or MCL1 at doses which are individually sub-lethal, we find that a combined BH3-mimetic approach targeting both pro-survival proteins enhances lethality and durably suppresses leukemic burden, regardless of TP53 mutation status. Our findings highlight the importance of employing sufficiently lethal treatment strategies to maximize outcomes for patients with TP53-mutant disease. In addition, our findings caution against use of sub-lethal BH3-mimetic drug regimens, which may enhance the risk of disease progression driven by emergent TP53 mutant clones.

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

Venetoclax inhibits the pro-survival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL, but with eventual loss of efficacy. A spectrum of sub-clonal genetic changes associated with venetoclax resistance have now been described. To fully understand clinical resistance to venetoclax, we combined single-cell short- and long‑read RNA‑sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired venetoclax resistance. These appear to be multi-layered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the pro-survival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but heterogeneous across and within individual patient's leukemias. Changes in the pro-apoptotic genes are notably uncommon, except for single cases with sub-clonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF‑kB activation which persisted in circulating cells during venetoclax therapy. We discovered that MCL1 could be a direct transcriptional target of NF‑kB. Both the switch to alternative pro-survival factors and NF‑kB activation largely dissipate following venetoclax discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade venetoclax-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent venetoclax resistance and provide a specific biological justification for the strategy of venetoclax discontinuation once maximal response is achieved rather than maintaining long-term selective pressure with the drug.