Highlights from Recent Cancer Literature

Multiplex Modeling of Cancer-Associated Variants

Weber and colleagues use the CRISPR/Cas9 system to multiplex functional genomic analyses. In models of intrahepatic cholangiocarcinoma and hepatocellular carcinoma, the authors perform an in vivo screen targeting a panel of tumor suppressor genes. Plasmid delivery to hepatocytes via hydrodynamic tail vein injection enabled in vivo screening of 10 single guide RNA sequences (sgRNA) in combination with oncogenic Kras^G12D and 18 sgRNAs in combination with CCl₄ treatment. High mutant read frequencies using next-generation sequencing in distinct tumor nodules suggest applications towards dissecting intratumoral heterogeneity and phylogenetic relationships among tumor foci. In addition, a multiplexed in vivo screen has the potential to assess the role for complex genetic interactions and tumor-tumor microenvironment interactions.

Weber J, Öllinger R, Friedrich M, Ehmer U, Barenboim M, Steiger K, et al. CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice. Proc Natl Acad Sci U S A 2015;112:13982–7.

The Cancer Driver from Within: Oncogenic RAB35 Drives AKT

To identify new regulators of the PI3K pathway, Wheeler and colleagues employed an RNAi screen using AKT phosphorylation as a read-out. They focused on genes not previously linked to the pathway and with somatic mutations in human cancers. The best represented functional group fulfilling these criteria were RAB GTPases. Loss of RAB35 suppressed AKT phosphorylation in response to receptor tyrosine kinase (RTK) signaling. Forced expression of active RAB35 was sufficient to activate PI3K/AKT in the absence of RTK stimulation. NIH3T3 cells carrying GTPase-activating mutations found in human tumors showed transformation and suppression of anoikis. The authors suggest that RAB35 drives constitutive endosomal signaling of PDGFRα to activate PI3K signaling. The emergence of RAB proteins as critical regulators of oncogenic signaling highlights the importance of endosomal traffic in human cancer.

Wheeler DB, Zoncu R, Root DE, Sabatini DM, Sawyers CL. Identification of an oncogenic RAB protein. Science 2015;350:211–7.

Microbiota Modulate Response to Immune Checkpoint Blockade

Although blockade of immune checkpoint pathways (CTLA-4 and the PD-1/PD-L1) shows durable activity against some melanomas, overall response rates are modest. The gut microbiota impacts systemic immune responses and immune responses following traditional cytotoxics. Two groups now demonstrate that the composition of gut microbiota influences efficacy of checkpoint blockade. Using 16S rRNA sequencing to profile specific bacteria in mouse models of melanoma, the groups identified microbial species correlated with antitumor responses. Subsequent gut reconstitution experiments with fecal transfer or direct administration of the microbial species could confer responses to anti-CTLA-4 and/or anti- PD-1/PD-L1 treatments. These reports open doors to 16S rRNA sequencing of patient stool samples as a novel biomarker and to gut microbial transplantation as a means to improve responses to immune checkpoint therapies.

Vétizou M, Pitt JM, Daillère R, Lepage P, Waldschmitt N, Flament C, et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science 2015;350:1079–84.

Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K, Earley ZM, et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti–PD-L1 efficacy. Science 2015;350:1084–9.

Unraveling Why Children Get Cancer

Over 100 cancer predisposition genes have been identified, providing insights into the genetics of tumor initiation. To define the prevalence of heritable mutations in children with cancer, Zhang and colleagues performed germline DNA sequencing from 1,120 such patients using a candidate-gene approach and report that 8.5% had a pathogenic mutation. Greater than half were in the TP53 tumor-suppressor gene, with evidence for somatic biallelic activation. Notably, a family history of cancer could only be uncovered in less than half of cases with a presumed inherited mutation. This study was biased towards analysis of the germline in patients with high-risk childhood cancers, so it may not accurately estimate the overall frequency of heritable mutations. Irrespective, these insights advocate for an awareness of cancer predisposition in the pediatric oncology clinic.

Zhang J, Walsh MF, Wu G, Edmonson MN, Gruber TA, Easton J. Germline mutations in predisposition genes in pediatric cancer. N Engl J Med 2015 Nov 18. doi: 10.1056/NEJMoa1508054. [Epub ahead of print].

PD-1 Inactivates NF-κB to Inhibit TIDCs in Ovarian Cancer

T-cell–dependent tumor immunity can be suppressed by the PD-1 (PDCD1):PD-L1 (CD274) immune signaling axis. Both human and murine tumor-infiltrating myeloid dendritic cells (TIDC) are implicated in immune escape and have elevated expression of PD-1. How PD-1 regulates innate immune cells remains to be defined. Karyampudi and colleagues examine the role of PD-1 in TIDC from mice with ovarian tumors. Expression of PD-1 on TIDC was associated with expression of the adaptor protein SHP-2 (PTPN11), which inhibited NF-κB activation. Although PD-1 blocked NF-κB–dependent cytokine release in a manner dependent on SHP-2, inhibition of NF-κB–mediated antigen presentation by PD-1 was independent of SHP-2. Thus PD-1 acts differently in innate immune cells versus adaptive immune cells. Further investigations into signaling pathways controlled by PD-1 might provide insights into immune escape in cancer.

Karyampudi L, Lamichhane P, Krempski J, Kalli KR, Behrens MD, Vargas DM et al. PD-1 blunts the function of ovarian tumor-infiltrating dendritic cells by inactivating NF-KappaB. Cancer Research; Published OnlineFirst November 13, 2015; doi: 10.1158/0008-5472.CAN-15-0748.

Modeling Lethal Metastatic Human Prostate Cancer

MYC activation and PTEN loss are common in lethal metastatic human prostate, suggesting roles in progression. Although prostate cancer develops in genetically engineered mouse models (GEMM) with engineered MYC overexpression and Pten deletion, these models only partially resemble lethal human disease. The Bieberich and DeMarzo labs used Hoxb13 regulatory elements to direct expression of MYC and deletion of mouse prostate secretory epithelium and a subset of basal cells, with the avoidance of androgen receptor-dependent control elements. These mice were highly penetrant for lethal metastatic castrate-resistant adenocarcinoma, possessed widespread genome copy number changes, and lacked neuroendocrine or sarcomatoid features common to other prostate cancer GEMMs. This novel prostate cancer GEMM should allow the testing of relevant prostate cancer therapies in an immunocompetent model, such as immunotherapies.

Hubbard GK, Mutton LN, Khalili M, McMullin RP, Hicks JL, Bianchi-Frias D, et al. Combined MYC activation and Pten loss are sufficient to create genomic instability and lethal metastatic prostate cancer. Cancer Research; Published OnlineFirst November 10, 2015; doi: 10.1158/0008-5472.CAN-14-3280.

Note: Breaking Advances are written by Cancer Research editors. Readers are encouraged to consult the articles referred to in each item for full details on the findings described.