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While immunotherapy has become standard-of-care for cutaneous melanoma patients, primary and acquired resistance prevent long-term benefits for about half of the late-stage patients. Pre-clinical models are essential to increase our understanding of the resistance mechanisms of melanomas, aiming to improve the efficacy of immunotherapy. Here, we present two novel syngeneic transplantable murine melanoma cell lines derived from the same primary tumor induced on BrafV600E Pten-/- mice: MeVa2.1 and MeVa2.2. Derivatives of these cell lines expressing the foreign antigen ovalbumin (dOVA) showed contrasting immune-mediated tumor control. MeVa2.2.dOVA melanomas were initially controlled in immune-competent hosts until variants grew out that had lost their antigens. By contrast, MeVa2.1.dOVA tumors were not controlled despite presenting the strong OVA antigen, as well as infiltration of tumor-reactive CD8+ T cells. MeVa2.1.dOVA displayed reduced sensitivity to T cell-mediated killing and growth inhibition in vitro by both IFN-γ and TNF-α. MeVa2.1.dOVA tumors were transiently controlled in vivo by either targeted therapy, adoptive T cell transfer, regulatory T cell depletion, or immune checkpoint blockade. MeVa2.1.dOVA could thus become a valuable melanoma model to evaluate novel immunotherapy combinations aiming to overcome immune resistance mechanisms.

Resistance to cancer immunotherapy continues to impair common clinical benefit. Here, we uncover an important role for Tuberous Sclerosis Complex 2 (TSC2) in determining tumor susceptibility to cytotoxic T lymphocytes (CTL) killing, bothin vitroandin vivo.TSC2-depleted tumor cells showed disrupted mTOR regulation upon CTL attack, which was associated with enhanced cell death. Tumor cells adapted to CTL attack by shifting their mTOR signaling balance toward increased mTORC2 activity to circumvent apoptosis and necroptosis. TSC2 critically protected tumor cells as its ablation strongly augmented tumor cell sensitivity to CTL attack. Mechanistically, TSC2 inactivation caused elevation of TRAIL receptors expression, cooperating with mTORC1-S6 signaling to induce tumor cell death. Clinically, we found a negative correlation betweenTSC2expression and TRAIL signaling in TCGA patient cohorts. Moreover, a lower TSC2 immune response signature was observed in melanomas from patients responding to immune checkpoint blockades. Our study uncovers a pivotal role for TSC2 in the cancer immune response by governing crosstalk between TSC2-mTOR and TRAIL signaling, aiding future therapeutic exploration of this pathway in immuno-oncology.

JMJD5, a Jumonji C domain-containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxia-induced cell proliferation. JMJD5 interacts directly with pyruvate kinase muscle isozyme (PKM)2 to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes hypoxia-inducible factor (HIF)-1α-mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2-HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α, is recruited to the hypoxia response element site in the lactate dehydrogenase A and PKM2 loci and mediates the recruitment of the latter two proteins. Our data uncover a mechanism whereby PKM2 can be regulated by factor-binding-induced homo/heterooligomeric restructuring, paving the way to cell metabolic reprogram.

Background Autophagy and molecular chaperones both regulate protein homeostasis and maintain important physiological functions. Atg7 (autophagy-related gene 7) and Hsp27 (heat shock protein 27) are involved in the regulation of neurodegeneration and aging. However, the genetic connection between Atg7 and Hsp27 is not known. Methods The appearances of the fly eyes from the different genetic interactions with or without polyglutamine toxicity were examined by light microscopy and scanning electronic microscopy. Immunofluorescence was used to check the effect of Atg7 and Hsp27 knockdown on the formation of autophagosomes. The lifespan of altered expression of Hsp27 or Atg7 and that of the combination of the two different gene expression were measured. Results We used the Drosophila eye as a model system to examine the epistatic relationship between Hsp27 and Atg7. We found that both genes are involved in normal eye development, and that overexpression of Atg7 could eliminate the need for Hsp27 but Hsp27 could not rescue Atg7 deficient phenotypes. Using a polyglutamine toxicity assay (41Q) to model neurodegeneration, we showed that both Atg7 and Hsp27 can suppress weak, toxic effect by 41Q, and that overexpression of Atg7 improves the worsened mosaic eyes by the knockdown of Hsp27 under 41Q. We also showed that overexpression of Atg7 extends lifespan and the knockdown of Atg7 or Hsp27 by RNAi reduces lifespan. RNAi-knockdown of Atg7 expression can block the extended lifespan phenotype by Hsp27 overexpression, and overexpression of Atg7 can extend lifespan even under Hsp27 knockdown by RNAi. Conclusions We propose that Atg7 acts downstream of Hsp27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila.

JMJD5, a JmjC-domain containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxia-induced cell proliferation. JMJD5 interacts directly with PKM2, pyruvate kinase M2, to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes HIF-1α-mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2-HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α are all recruited to the HRE (hypoxia response element) site in the LDHA and PKM2 loci, and mediates the recruitment of the latter two proteins. Our data uncovers a new mechanism whereby PKM2 can be regulated by factor-binding induced homo/hetero-oligomeric restructuring, paving the way to cell metabolic reprogram. Citation Format: Hung-Jung Wang, Ya-Ju Hsieh, Wen-Chi Cheng, Chun-Pu Lin, Yu-Shan Lin, So-Fang Yang, Chung-Ching Chen, Yoshihiro Izumiya, Jau-Song Yu, Hsing-Jien Kung, Wen-Ching Wang. JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1α-mediated glucose metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3359. doi:10.1158/1538-7445.AM2014-3359