Summary Combination immune checkpoint blockade has demonstrated promising benefit in lung cancer, but predictors of response to combination therapy are unknown. Using whole exome sequencing to examine non-small cell lung cancer (NSCLC) treated with PD-1 plus CTLA-4 blockade, we found that high tumor mutation burden (TMB) predicted improved objective response, durable benefit, and progression-free survival. TMB was independent of PD-L1 expression and the strongest feature associated with efficacy in multivariable analysis. The low response rate in TMB low NSCLCs demonstrates that combination immunotherapy does not overcome the negative predictive impact of low TMB. This study demonstrates the association between TMB and benefit to combination immunotherapy in NSCLC. TMB should be incorporated in future trials examining PD-(L)1 with CTLA-4 blockade in NSCLC.

The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic "knock-in" strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1␣ with HIF-2␣ results in expanded expression of HIF-2␣-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2␣, but not HIF-1␣, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2␣ KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2␣ severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2␣-specific target gene and indicate that HIF-2␣ can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2␣'s tumor promoting activity.[Keywords: HIF; hypoxia; HIF-2␣; Oct-4; VEGF; TGF-␣; stem cells; cancer] Supplemental material is available at http://www.genesdev.org.

Mammalian cells are believed to have a cell-intrinsic ability to increase glucose metabolism in response to hypoxia. Here we show that the ability of hematopoietic cells to up-regulate anaerobic glycolysis in response to hypoxia is dependent on receptor-mediated signal transduction. In the absence of growth factor signaling, hematopoietic cells fail to express hypoxia-inducible transcription factor (Hif-1␣) mRNA. Growth factor-deprived hematopoietic cells do not engage in glucose-dependent anabolic synthesis and neither express Hif-1␣ mRNA nor require HIF-1␣ protein to regulate cell survival in response to hypoxia. However, HIF-1␣ is adaptive for the survival of growth factor-stimulated cells, as suppression of HIF-1␣ results in death when growing cells are exposed to hypoxia. Growth factor-dependent HIF-1␣ expression reprograms the intracellular fate of glucose, resulting in decreased glucose-dependent anabolic synthesis and increased lactate production, an effect that is enhanced when HIF-1␣ protein is stabilized by hypoxia. Together, these data suggest that HIF-1␣ contributes to the regulation of growth factor-stimulated glucose metabolism even in the absence of hypoxia.[Keywords: HIF-1␣; hypoxia; growth factor signaling; glucose metabolism; cell survival] Supplemental material is available at http://www.genesdev.org.

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Hypoxia-inducible factors (HIF) are essential transcriptional regulators that mediate adaptation to hypoxic stress in rapidly growing tissues such as tumors. HIF activity is regulated by hypoxic stabilization of the related HIF-1A and HIF-2A subunits, which are frequently overexpressed in cancer cells. To assess the relative tumor-promoting functions of HIF-1A and HIF-2A directly, we replaced HIF-1A expression with HIF-2A by creating a novel ''knock-in'' allele at the Hif-1a locus through homologous recombination in primary murine embryonic stem cells. Compared with controls, s.c. teratomas derived from knock-in embryonic stem cells were larger and more proliferative, had increased microvessel density, and exhibited increased expression of vascular endothelial growth factor, transforming growth factor-A, and cyclin D1. These and other data indicate that HIF-2A promotes tumor growth more effectively than HIF-1A in multiple contexts. (Cancer Res 2005; 65(6): 2277-86)