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Background: Docetaxel has immunostimulatory effects that may promote an immunoresponsive prostate tumour microenvironment, providing a rationale for combination with nivolumab (programmed death-1 inhibitor) for metastatic castration-resistant prostate cancer (mCRPC)., Methods: In the non-randomised, multicohort, global phase II CheckMate 9KD trial, 84 patients with chemotherapy-naive mCRPC, ongoing androgen deprivation therapy and ≤2 prior novel hormonal therapies (NHTs) received nivolumab 360 mg and docetaxel 75 mg/m 2 every 3 weeks with prednisone 5 mg twice daily (≤10 cycles) and then nivolumab 480 mg every 4 weeks (≤2 years). The co-primary end-points were objective response rate (ORR) and prostate-specific antigen response rate (PSA 50 -RR; ≥50% decrease from baseline)., Results: The confirmed ORR (95% confidence interval [CI]) was 40.0% (25.7-55.7), and the confirmed PSA 50 -RR (95% CI) was 46.9% (35.7-58.3). The median (95% CI) radiographic progression-free survival (rPFS) and overall survival (OS) were 9.0 (8.0-11.6) and 18.2 (14.6-20.7) months, respectively. In subpopulations with versus without prior NHT, the ORR was 38.7% versus 42.9%, the PSA 50 -RR was 39.6% versus 60.7%, the median rPFS was 8.5 versus 12.0 months and the median OS was 16.2 months versus not reached. Homologous recombination deficiency status or tumour mutational burden did not appear to impact efficacy. The most common any-grade and grade 3-4 treatment-related adverse events were fatigue (39.3%) and neutropenia (16.7%), respectively. Three treatment-related deaths occurred (1 pneumonitis related to nivolumab; 2 pneumonias related to docetaxel)., Conclusions: Nivolumab plus docetaxel has clinical activity in patients with chemotherapy-naïve mCRPC. Safety was consistent with the individual components. These results support further investigation in the ongoing phase III CheckMate 7DX trial. CLINICALTRIALS., Gov Registration: NCT03338790., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: To define dominant molecular and cellular features associated with PD-1/PD-L1 blockade resistance in metastatic urothelial cancer., Experimental Design: We pursued an unbiased approach using bulk RNA sequencing data from two clinical trials to discover (IMvigor 210) and validate (CheckMate 275) pretreatment molecular features associated with resistance to PD-1/PD-L1 blockade in metastatic urothelial cancer. We then generated single-cell RNA sequencing (scRNA-seq) data from muscle-invasive bladder cancer specimens to dissect the cellular composition underlying the identified gene signatures., Results: We identified an adaptive immune response gene signature associated with response and a protumorigenic inflammation gene signature associated with resistance to PD-1/PD-L1 blockade. The adaptive immune response:protumorigenic inflammation signature expression ratio, coined the 2IR score, best correlated with clinical outcomes, and was externally validated. Mapping these bulk gene signatures onto scRNA-seq data uncovered their underlying cellular diversity, with prominent expression of the protumorigenic inflammation signature by myeloid phagocytic cells. However, heterogeneity in expression of adaptive immune and protumorigenic inflammation genes was observed among single myeloid phagocytic cells, quantified as the myeloid single cell immune:protumorigenic inflammation ratio (M sc 2IR) score. Single myeloid phagocytic cells with low M sc 2IR scores demonstrated upregulation of proinflammatory cytokines/chemokines and downregulation of antigen presentation genes, were unrelated to M1 versus M2 polarization, and were enriched in pretreatment blood samples from patients with PD-L1 blockade-resistant metastatic urothelial cancer., Conclusions: The balance of adaptive immunity and protumorigenic inflammation in individual tumor microenvironments is associated with PD-1/PD-L1 resistance in urothelial cancer with the latter linked to a proinflammatory cellular state of myeloid phagocytic cells detectable in tumor and blood. See related commentary by Drake, p. 4139 ., (©2021 American Association for Cancer Research.)

Background: The role of adjuvant treatment in high-risk muscle-invasive urothelial carcinoma after radical surgery is not clear., Methods: In a phase 3, multicenter, double-blind, randomized, controlled trial, we assigned patients with muscle-invasive urothelial carcinoma who had undergone radical surgery to receive, in a 1:1 ratio, either nivolumab (240 mg intravenously) or placebo every 2 weeks for up to 1 year. Neoadjuvant cisplatin-based chemotherapy before trial entry was allowed. The primary end points were disease-free survival among all the patients (intention-to-treat population) and among patients with a tumor programmed death ligand 1 (PD-L1) expression level of 1% or more. Survival free from recurrence outside the urothelial tract was a secondary end point., Results: A total of 353 patients were assigned to receive nivolumab and 356 to receive placebo. The median disease-free survival in the intention-to-treat population was 20.8 months (95% confidence interval [CI], 16.5 to 27.6) with nivolumab and 10.8 months (95% CI, 8.3 to 13.9) with placebo. The percentage of patients who were alive and disease-free at 6 months was 74.9% with nivolumab and 60.3% with placebo (hazard ratio for disease recurrence or death, 0.70; 98.22% CI, 0.55 to 0.90; P<0.001). Among patients with a PD-L1 expression level of 1% or more, the percentage of patients was 74.5% and 55.7%, respectively (hazard ratio, 0.55; 98.72% CI, 0.35 to 0.85; P<0.001). The median survival free from recurrence outside the urothelial tract in the intention-to-treat population was 22.9 months (95% CI, 19.2 to 33.4) with nivolumab and 13.7 months (95% CI, 8.4 to 20.3) with placebo. The percentage of patients who were alive and free from recurrence outside the urothelial tract at 6 months was 77.0% with nivolumab and 62.7% with placebo (hazard ratio for recurrence outside the urothelial tract or death, 0.72; 95% CI, 0.59 to 0.89). Among patients with a PD-L1 expression level of 1% or more, the percentage of patients was 75.3% and 56.7%, respectively (hazard ratio, 0.55; 95% CI, 0.39 to 0.79). Treatment-related adverse events of grade 3 or higher occurred in 17.9% of the nivolumab group and 7.2% of the placebo group. Two treatment-related deaths due to pneumonitis were noted in the nivolumab group., Conclusions: In this trial involving patients with high-risk muscle-invasive urothelial carcinoma who had undergone radical surgery, disease-free survival was longer with adjuvant nivolumab than with placebo in the intention-to-treat population and among patients with a PD-L1 expression level of 1% or more. (Funded by Bristol Myers Squibb and Ono Pharmaceutical; CheckMate 274 ClinicalTrials.gov number, NCT02632409.)., (Copyright © 2021 Massachusetts Medical Society.)

In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumour suppressor STK11 (also known as LKB1) encoding the kinase LKB1 result in aggressive tumours prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analysed tumour metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KRAS/LKB1 co-mutant tumours. KRAS/LKB1 co-mutant cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme glutamine-fructose-6-phosphate transaminase [isomerizing] 2 (GFPT2). GFPT2 inhibition selectively reduced KRAS/LKB1 co-mutant tumour cell growth in culture, xenografts and genetically modified mice. Our results define a new metabolic vulnerability in KRAS/LKB1 co-mutant tumours and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.

Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies have demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. Here, we demonstrate that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, because of a reduction in intracellular guanine nucleotides. AG2037 treatment provokes both mTORC1 inhibition and robust tumor growth suppression in mice bearing non-small-cell lung cancer (NSCLC) xenografts. These results indicate that alterations in purine nucleotide availability affect mTORC1 activity and suggest that inhibition of mTORC1 contributes to the therapeutic effects of purine biosynthesis inhibitors., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metabolic reprogramming by oncogenic signals promotes cancer initiation and progression. The oncogene KRAS and tumour suppressor STK11, which encodes the kinase LKB1, regulate metabolism and are frequently mutated in non-small-cell lung cancer (NSCLC). Concurrent occurrence of oncogenic KRAS and loss of LKB1 (KL) in cells specifies aggressive oncological behaviour. Here we show that human KL cells and tumours share metabolomic signatures of perturbed nitrogen handling. KL cells express the urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1), which produces carbamoyl phosphate in the mitochondria from ammonia and bicarbonate, initiating nitrogen disposal. Transcription of CPS1 is suppressed by LKB1 through AMPK, and CPS1 expression correlates inversely with LKB1 in human NSCLC. Silencing CPS1 in KL cells induces cell death and reduces tumour growth. Notably, cell death results from pyrimidine depletion rather than ammonia toxicity, as CPS1 enables an unconventional pathway of nitrogen flow from ammonia into pyrimidines. CPS1 loss reduces the pyrimidine to purine ratio, compromises S-phase progression and induces DNA-polymerase stalling and DNA damage. Exogenous pyrimidines reverse DNA damage and rescue growth. The data indicate that the KL oncological genotype imposes a metabolic vulnerability related to a dependence on a cross-compartmental pathway of pyrimidine metabolism in an aggressive subset of NSCLC.

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1-3 follows a sequential ordered Bi-Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in k cat for PKN1 and PKN2, and a decrease in peptide K M for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (K i <10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors., (© 2014 The author(s).)

Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here, we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1 and that elevated MCT1 levels are manifest in premalignant and neoplastic Eμ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, reductions in glucose transport, and in levels of ATP, NADPH, and ultimately, glutathione (GSH). Reductions in GSH then lead to increases in hydrogen peroxide, mitochondrial damage, and ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies.

PKN3 is an AGC-family protein kinase implicated in growth of metastatic prostate cancer cells with phosphoinositide 3-kinase pathway deregulation. The molecular mechanism, however, by which PKN3 contributes to malignant growth and tumorigenesis is not well understood. Using orthotopic mouse tumor models, we now show that inducible knockdown of PKN3 protein not only blocks metastasis, but also impairs primary prostate and breast tumor growth. Correspondingly, overexpression of exogenous PKN3 in breast cancer cells further increases their malignant behavior and invasiveness in-vitro. Mechanistically, we demonstrate that PKN3 physically interacts with Rho-family GTPases, and preferentially with RhoC, a known mediator of tumor invasion and metastasis in epithelial cancers. Likewise, RhoC predominantly associates with PKN3 compared to its closely related PKN family members. Unlike the majority of Rho GTPases and PKN molecules, which are ubiquitously expressed, both PKN3 and RhoC show limited expression in normal tissues and become upregulated in late-stage malignancies. Since PKN3 catalytic activity is increased in the presence of Rho GTPases, the co-expression and preferential interaction of PKN3 and RhoC in tumor cells are functionally relevant. Our findings provide novel insight into the regulation and function of PKN3 and suggest that the PKN3-RhoC complex represents an attractive therapeutic target in late-stage malignancies., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)