Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

To understand how genomic heterogeneity of glioblastoma contributes to the poor response to therapy characteristic of this disease, we performed DNA and RNA sequencing on GBM tumor samples and the neurospheres and orthotopic xenograft models derived from them. We used the resulting data set to show that somatic driver alterations including single nucleotide variants, focal DNA alterations, and oncogene amplification on extrachromosomal DNA (ecDNA) elements were in majority propagated from tumor to model systems. In several instances, ecDNAs and chromosomal alterations demonstrated divergent inheritance patterns and clonal selection dynamics during cell culture and xenografting. We infer that ecDNA inherited unevenly between offspring cells, a characteristic that affects the oncogenic potential of cells with more or fewer ecDNAs. Longitudinal patient tumor profiling found that oncogenic ecDNAs are frequently retained throughout the course of disease. Our analysis shows that extrachromosomal elements allow rapid increase of genomic heterogeneity during glioblastoma evolution, independent of chromosomal DNA alterations.

Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We previously identified a subset of cancers harboring homozygous deletion of the glycolytic enzyme Enolase (ENO1) with exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small molecule Enolase inhibitor, POMHEX, can selectively kill ENO1 -deleted glioma cells at low nanomolar concentrations and eradicate intracranial orthotopic ENO1 -deleted tumors in mice at doses well-tolerated in non-human primates. Our data provide in vivo proof-of-principal for the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential across a range of therapeutic settings.

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Abstract Ibrutinib is highly efficacious and used at 420 mg/d for treatment of chronic lymphocytic leukemia (CLL). We previously demonstrated a decline in Bruton’s tyrosine kinase (BTK) protein levels in CLL cells after 1 cycle of ibrutinib, suggesting ibrutinib dose could be lowered after the first cycle without loss of biological effect. To test this postulate, a pilot study (NCT02801578) was designed to systematically reduce ibrutinib dosing within the same patient with CLL over the course of three 28-day cycles. After an initial cycle of 420 mg/d, the dose was reduced to 280 mg/d in cycle 2, and then to 140 mg/d in cycle 3. Eleven patients began study treatment, and 9 completed the 3 cycles. Plasma and intracellular pharmacokinetics (PK), BTK occupancy, and pharmacodynamic (PD) response at different doses of ibrutinib were compared. Plasma and intracellular levels of ibrutinib were dose-dependent, and even the lowest dose was sufficient to occupy, on average, more than 95% of BTK protein. In concert, BTK downstream signaling inhibition was maintained with 140 mg/d ibrutinib in cycle 3, and there were comparable reductions in total and phospho-BTK (Tyr223) protein levels across 3 cycles. Reductions of plasma chemokine CCL3 and CCL4 levels, considered to be biomarkers of ibrutinib response, were similar during the 3 cycles. These PK/PD data demonstrate that after 1 cycle of ibrutinib at the standard 420 mg/d dose, the dose can be reduced without losing biological activity. Clinical efficacy of lower doses needs to be systematically evaluated. Such dose reductions would lower drug cost, lessen untoward toxicity, and facilitate rationale-based combinations. This trial was registered at www.clinicaltrials.gov as #NCT02801578.

Cytochromes P450 cam and P450 BM3 oxidize α-and β-thujone into multiple products, including 7-hydroxy-α-(or β-)thujone, 7,8-dehydro-α-(or β-)thujone, 4-hydroxy-α-(or β-)thujone, 2-hydroxy α-(or β-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 ± 0.3 × 10 10 s −1 to 12.5 ± 3 × 10 10 s −1 for trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[ 2 H]-α-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450 cam active site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of α-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450 cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate-and enzyme-dependent.The regio-and stereoselective hydroxylation of hydrocarbons by cytochrome P450 enzymes is a chemically demanding but physiologically important reaction. Hydroxylation is required, for example, for the conversion of cholesterol into a panoply of mammalian sterol hormones and of arachidonic acid into vasodilator and vasoconstrictor signaling molecules (1,2). The mechanism of carbon hydroxylation has been thought for three decades to involve hydrogen abstraction by the catalytic ferryl species followed by recombination of the resulting substrate carbon radical with the equivalent of an iron-bound hydroxyl radical (3,4). The ferryl species involved in this reaction is generated by reductive activation of molecular oxygen at the iron † This work was supported by grant GM25515 from the National Institutes of Health.

Cyclic and acyclic nitroaryl phosphoramide mustard analogues were activated by E. coli nitroreductase, an enzyme explored in GDEPT. The more active acyclic 4-nitrobenzyl phosphoramide mustard (7) showed 167 500x selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC(50) as low as 0.4 nM. This is about 100x more active and 27x more selective than CB1954 (1). The superior activity was attributed to its better substrate activity (k(cat)/K(m) 19x better than 1) and/or excellent cytotoxicity of phosphoramide mustard released.

Highlights d PGD is a top hit in a loss-of-function genetics screen in OXPHOS-deficient cancer d OXPHOS-deficient cells depend on PGD in vitro and in vivo d PGD inhibition affects glycolysis, reductive carboxylation, and redox homeostasis d Pharmacological inhibition of OXPHOS renders PGD dependent