KRASis the most frequently mutatedrasfamily member in lung carcinomas 1,2 , whereasHRASmutations are common in tumours from stratified epithelia such as bladder or skin (www.sanger.ac.uk/genetics/CGP/cosmic/). Using a mouse model (HrasKI) 3 in which theHrascoding sequence was inserted into theKraslocus, we demonstrate that specificity forKrasmutations in lung andHras mutations in skin tumours is determined by local regulatory elements in the targetrasgenes. We further show that, whileKras-4Ais dispensable for mouse development 4,5 , it is necessary both for lung carcinogenesisin vivoand for the previously reported 6,7 inhibitory effect of wild-type (WT)Krason the transforming properties of the mutant allele. Kras-4A expression is detected in a sub-population of normal lung epithelial cells, but at very low levels in lung tumours, suggesting a role in tumour initiation rather than in tumour maintenance. The two Kras isoforms undergo different post-translational modifications 8 , therefore these findings can have important implications for the design of therapeutic strategies for inhibiting oncogenic Kras activity in the prevention and treatment of cancer.Kras deficiency in mice leads to embryonic lethality 4,5 , thus it has not been possible to determine whether the selection for Kras mutations in lung tumours reflects a specific oncogenic function that is required for lung carcinogenesis and cannot be compensated for by mutant Hras or Nras. The HrasKI mouse provides a viable model that completely lacks Kras expression 3 (Fig. 1), but carries a "knock-in" Hras cDNA expressed under the control of Kras regulatory elements. We reasoned that HrasKI mice should be resistant to lung tumour development if Kras protein is essential for lung carcinogenesis. To control for the possibility of structural alterations affecting experimental outcomes, we used KrasKI mice in which the coding sequence corresponding to the Kras-4B isoform was knocked back into the Kras locus in the exact manner used to generate the HrasKI mouse 3 . HrasKI mice expressed elevated levels of Hras protein, consistent with the increase in Hras gene dosage from two copies to four in these animals ( Fig. 1). KrasKI mice express similar levels of Kras protein as WT mice, as expected since Kras-4B is the major splice isoform 9,10 . Levels of Akt and Erk activation in these animals were similar to WT mice (Fig. 1). There was also no difference in levels of p38α, recently shown to affect ras mediated lung carcinogenesis 11 . The elevated level of Hras We further pursued the specificity of ras gene mutations in lung and skin tumours by inducing both tumour types in the same HrasKI homozygous mice. A single dose of urethane was injected intraperitoneally to induce lung tumours, followed by a single topical treatment on the skin with urethane and subsequent biweekly application of tetradecanoyl-phorbol acetate (TPA) to induce papilloma development. All lung tumours (n = 28) analyzed have the CAA>CTA mutation in the HrasKI allele. Of the papillomas (n = ...

Background Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFβ signaling. Methods To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFβ or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses. Results We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFβ monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFβ, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFβ antibody administration attenuates these effects. We show that α-TGFβ acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFβ acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery. Conclusions We show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFβ-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFβ combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFβ/α-PD-1 combination therapy (NCT02947165). Electronic supplementary material The online version of this article (10.1186/s40425-018-0493-9) contains supplementary material, which is available to authorized users.

) mice develop increased papilloma numbers and show decreased carcinoma latency time in comparison with controls after skin treatment with dimethyl benzanthracene (DMBA) and tetradecanoyl-phorbol acetate (TPA). H-ras mutation is normally a hallmark of DMBA-TPA-induced skin tumors, but 70% of carcinomas from Pten +/− mice do not exhibit this mutation, and in all cases have lost the wild-type Pten allele. Tumors that retain the Pten wild-type allele also have H-ras mutations, indicating that activation of Hras and complete loss of Pten are mutually exclusive events in skin carcinomas. Mitogen-activated protein kinase (MAPK) is consistently activated in the tumors with H-ras mutations, but is strongly down-regulated in Pten −/− tumors, suggesting that this pathway is dispensable for skin carcinoma formation. These data have important implications in designing individual therapeutic strategies for the treatment of cancer.