Reduced mitochondrial transcription sensitizes acute myeloid leukemia cells to BCL-2 inhibition

Reviewer #1 (Public Review):

This study exploits novel agent (IMT) that inhibits mitochondrial activity in combination with venetoclax. While the concept is not novel, the agent is novel (inhibitor of the mitochondrial RNA polymerase, described in Nature in other tumor models), and quest for safe mitochondrial inhibitors is highly warranted. The strength is in vivo activity data shown in CLDX and in one of the two AML PDX models tested, and the apparent safety of the combination. However, the impact on survival is impressive in CLDX but not in PDX, and unclear why Ven-sensitive PDX is resistant to combination (opposite what cell line data show). The paper is lacking mechanistic data beyond Seahorse and standard apoptosis assays, and even transcriptome analysis from PDX cells is poorly analyzed. There is no real evidence that this agent overcome Ven resistance, which could be done for example in primary AML cells. Finally, no on-target pharmacodynamic endpoints are measured in vivo to support the activity of the compound on mitochondrial activity at the doses used (which are safe). These multiple weaknesses significantly reduce my enthusiasm for this manuscript.

The cell line data show additive/synergistic effects of IMT and Ven on cell viability in p53-WT cells. However, no mechanisms of synergy beyond OCR are shown, which is a missed opportunity.

No data are shown in primary AML cells in vitro. This could address venetoclax-resistant AML cells with distinct genomic profiles.

The in vivo CLDX model (MV4;11) data is quite impressive, showing reduction of tumor burden and meaningful extension of survival in combination cohort. It is unclear why venetoclax used at highest dose normally sued in vivo (100mg/kg) did not show any impact on survival in this Ven-sensitive model. It is disappointing that no biomarkers of mitochondrial activity (for example, simple pAMPK, or levels of mitochondrial subunits) are shown to support on-target pharmacodynamic activity. However, efficacy in human PDX is less impressive, for example in Fig 6C the combination has extended survival from 96 to 112 days, possibly due to early stopping of treatment (around day 30); and no extension of survival is seen in another PDX in Fig 7. Still, this is indicative of combinatorial activity in TP53-mutant PDX. There is however discrepancy with in vitro studies that show no impact of combination in TP53 mutant cells and synergy in TP53-wt cells, and the opposite findings in vivo, which is not explained. Overall, the activity of the combination is modest. The safety is encouraging, but again, no pharmacodynamic measurements are shown to support that IMT at least partially inhibited mitochondrial activity in AML cells.

In Discussion the statement that inhibition of POLRMT can overcome venetoclax resistance is not supported by the data, as no additive effects are seen in vitro in TP53 mutant cells, and no other resistant models (such as primary AML cells) are tested. In vivo as stated above there is some activity in TP53 mutant PDX but this alone cannot be sued to justify this strong statement. Also, the sentence that "...we were able to reduce the tumor burden in all (cell- and patient-derived) xenografted mice treated with a combination of IMT and venetoclax" is not supported by data in Fig 7.

Reviewer #2 (Public Review):

Summary:

The manuscript by Arabanian and colleagues presents studies showing how inhibition of mitochondrial transcription and replication with a novel inhibitor of the mitochondrial polymerase, IMT, can promote AML cell death in combination with the Bcl2 inhibitor venetoclax. They further show that this combinatorial efficacy is evident in vivo in both the AML cell line MV411 and in a PDX model. Given the multiple studies showing the importance of Oxphos in maintaining AML cell survival, the current studies provide an additional strategy to inhibit Oxphos and thus improve the therapeutic management of AML.

Strengths:

A novel aspect of this work is that IMT is a new class of mitochondrial inhibitor that acts by inhibiting the mitochondrial polymerase. In addition, the demonstration of therapeutic efficacy both in vitro and in vivo (including with PDX), together with some data showing minimal toxicity, adds to the impact of this work. Their overall conclusion that IMT increases the potency of Vex in treating AMLs is supported.

Weaknesses:

There are several deficiencies that should be addressed to substantiate the rigor and impact of this study. Of most importance, they need to show that IMT actually inhibits the mitochondrial polymerase in AML cells, and there are additional concerns with their models that if addressed would improve the ability of IMT to be developed clinically.