Predictors of responses to immune checkpoint blockade in advanced melanoma.

Immune checkpoint blockers (ICB) have become pivotal therapies in the clinical armamentarium against metastatic melanoma (MMel). Given the frequency of immune related adverse events and increasing use of ICB, predictors of response to CTLA-4 and/or PD-1 blockade represent unmet clinical needs. Using a systems biology-based approach to an assessment of 779 paired blood and tumor markers in 37 stage III MMel patients, we analyzed association between blood immune parameters and the functional immune reactivity of tumor-infiltrating cells after ex vivo exposure to ICB. Based on this assay, we retrospectively observed, in eight cohorts enrolling 190 MMel patients treated with ipilimumab, that PD-L1 expression on peripheral T cells was prognostic on overall and progression-free survival. Moreover, detectable CD137 on circulating CD8+ T cells was associated with the disease-free status of resected stage III MMel patients after adjuvant ipilimumab + nivolumab (but not nivolumab alone). These biomarkers should be validated in prospective trials in MMel.The clinical management of metastatic melanoma requires predictors of the response to checkpoint blockade. Here, the authors use immunological assays to identify potential prognostic/predictive biomarkers in circulating blood cells and in tumor-infiltrating lymphocytes from patients with resected stage III melanoma.

Co-amplified genes at 8p12 and 11q13 in breast tumors cooperate with two major pathways in oncogenesis.

Co-amplification at chromosomes 8p11-8p12 and 11q12-11q14 occurs often in breast tumors, suggesting possible cooperation between genes in these regions in oncogenesis. We used high-resolution array comparative genomic hybridization (array CGH) to map the minimal amplified regions. The 8p and 11q amplicons are complex and consist of at least four amplicon cores at each site. Candidate oncogenes mapping to these regions were identified by combining copy number and RNA and protein expression analyses. These studies also suggested that CCND1 at 11q13 induced expression of ZNF703 mapping at 8p12, which was subsequently shown to be mediated by the Rb/E2F pathway. Nine candidate oncogenes from 8p12 and four from 11q13 were further evaluated for oncogenic function. None of the genes individually promoted colony formation in soft agar or collaborated with each other functionally. On the other hand, FGFR1 and DDHD2 at 8p12 cooperated functionally with MYC, whereas CCND1 and ZNF703 cooperated with a dominant negative form of TP53. These observations highlight the complexity and functional consequences of the genomic rearrangements that occur in these breast cancer amplicons, including transcriptional cross-talk between genes in the 8p and 11q amplicons, as well as their cooperation with major pathways of tumorigenesis.

Functional analysis and intracellular localization of p53 modified by SUMO-1.

p53 tumor suppressor is a subject of several post-translational modifications, including phosphorylation, ubiquitination and acetylation, which regulate p53 function. A new covalent modification of p53 at lysine 386 by SUMO-1 was recently identified. To elucidate the function of sumoylated p53, we compared the properties of wild type p53 and sumoylation-deficient p53 mutant, K386R. No differences were found between wild type p53 and K386R mutant of p53 in transactivation or growth suppression assays. Moreover, overexpression of SUMO-1 has no effect on p53-regulated transcription. Biochemical fractionation showed that sumoylated p53 is localized in the nucleus and is tightly bound to chromatin structures. p53 and SUMO-1 co-localized in PML nuclear bodies in 293 cells and the nucleoli in MCF7 and HT1080 cells. However, sumoylation-deficient p53 mutant showed a similar pattern of intranuclear localization, suggesting that SUMO-1 does not target p53 to subnuclear structures. These data indicate that SUMO-1 modification of p53 at lysine 386 may not be essential for p53's cellular localization, transcriptional activation, or growth regulation.

Structure and regulation of the mouse ing1 gene. Three alternative transcripts encode two phd finger proteins that have opposite effects on p53 function.

The human ING1 gene encodes nuclear protein p33(ING1), previously shown to cooperate with p53 in cell growth control (Garkavtsev, I., Grigorian, I. A., Ossovskaya, V. S., Chernov, M. V., Chumakov, P. M., and Gudkov, A. V. (1998) Nature 391, 295-298). p33(ING1) belongs to a small family of proteins from human, mouse, and yeast of approximately the same size that show significant similarity to one another within the C-terminal PHD finger domain and also contain an additional N-terminal region with subtle but reliably detectable sequence conservation. Mouse ing1 is transcribed from three differently regulated promoters localized within a 4-kilobase pair region of genomic DNA. The resulting transcripts share a long common region encoded by a common exon and differ in their 5'-exon sequences. Two transcripts are translated into the same protein of 185 amino acids, the mouse equivalent of the human p33(ING1), while the third transcript encodes a longer protein that has 94 additional N-terminal amino acids. Overexpression of the longer protein interferes with the accumulation of p53 protein and activation of p53-responsive promoters after DNA damage. Between the two products of ing1, only the longer one forms a complex with p53 detectable by immunoprecipitation. These results indicate that a single gene, ing1, encodes both p53-suppressing and p53-activating proteins that are regulated by alternative promoters.

Suppression of apoptosis by bcl-2 does not prevent p53-mediated control of experimental metastasis and anchorage dependence.

Mutations in the p53 tumor suppressor gene are frequently associated with the metastatic stage of tumor progression. Inactivation of p53 was shown to promote metastasis under experimental conditions. To determine the p53 functions that are involved in the control of tumor metastasis, we compared properties of three types of transformed mouse fibroblasts: with intact p53, with p53-mediated apoptosis suppressed by bcl-2 and with p53 inactivated by dominant negative mutants. Although expression of bcl-2 blocked apoptosis in detached cells and increased tumor cell survival in the blood circulation, it was insufficient to affect the ability of p53 to cause cell cycle arrest in detached cells and suppress experimental metastasis. For the suppression of metastasis complete inactivation of p53 was required. We conclude that the apoptotic function of p53 is dispensable for the p53-dependent suppression of experimental metastasis that is presumably achieved by controlling anchorage dependence. These data provide a possible explanation to dramatic differences in values of bcl-2 and mutant p53 as prognostic markers in human cancer.