Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition.

Mammalian target of rapamycin (mTOR) regulates cellular processes important for progression of human cancer. RAD001 (everolimus), an mTORC1 (mTOR/raptor) inhibitor, has broad antitumor activity in preclinical models and cancer patients. Although most tumor lines are RAD001 sensitive, some are not. Selective mTORC1 inhibition can elicit increased AKT S473 phosphorylation, involving insulin receptor substrate 1, which is suggested to potentially attenuate effects on tumor cell proliferation and viability. Rictor may also play a role because rictor kinase complexes (including mTOR/rictor) regulate AKT S473 phosphorylation. The role of raptor and rictor in the in vitro response of human cancer cells to RAD001 was investigated. Using a large panel of cell lines representing different tumor histotypes, the basal phosphorylation of AKT S473 and some AKT substrates was found to correlate with the antiproliferative response to RAD001. In contrast, increased AKT S473 phosphorylation induced by RAD001 did not correlate. Similar increases in AKT phosphorylation occurred following raptor depletion using siRNA. Strikingly, rictor down-regulation attenuated AKT S473 phosphorylation induced by mTORC1 inhibition. Further analyses showed no relationship between modulation of AKT phosphorylation on S473 and T308 and AKT substrate phosphorylation patterns. Using a dual pan-class I phosphatidylinositol 3-kinase/mTOR catalytic inhibitor (NVP-BEZ235), currently in phase I trials, concomitant targeting of these kinases inhibited AKT S473 phosphorylation, eliciting more profound cellular responses than mTORC1 inhibition alone. However, reduced cell viability could not be predicted from biochemical or cellular responses to mTORC1 inhibitors. These data could have implications for the clinical application of phosphatidylinositol 3-kinase/mTOR inhibitors

Comprehensive mapping of p53 pathway alterations in sarcomas reveals an apparent role for MDM2 SNP309 in sarcomagenesis

Re-activation of p53 tumour suppressor activity in diseases such as soft tissue sarcomas is considered an attractive means of targeted therapy. To assess the pattern of mutations affecting the p53 pathway, we have comprehensively mapped mutational events in a panel of 192 bone and soft-tissue sarcomas. These include TP53 and CDKN2A mutational and SNP status, MDM2 and MDM4 amplification and MDM2 SNP309 status. Overall, we found an inverse relationship between MDM2 amplification and TP53 mutations in our samples. Although CDKN2A exon and gene deletions were observed, ARF was found to be predominantly wild-type. Alternatively, a high rate of point mutations in TP53 was observed in leiomyosarcoma and osteosarcoma. Our data show the expected high level of MDM2 amplification in well- and de-differentiated liposarcomas, as well subtype specific patterns. Similarly, MDM4 was amplified in a subtype specific manner. Notably, MDM2 and MDM4 amplification events were found to be frequently associated. We have also analysed the risk allele frequencies for MDM2 SNP309, and show that homozygosity for the G SNP was strongly associated with both liposarcomas and MDM2 amplification. Moreover, our data on a set of tumour-matched normal controls indicates a clear directional progression of the MDM2 SNP309 G allele in tumour samples. In summary, our data suggest that at least 70% of sarcomas sustain some type of genetic alterations in the p53 pathway, of which most impinge on either MDM2 or MDM4. We propose, therefore, that these tumour types should be suitable candidates for trials of MDM2 antagonists

Cancer lineage-specific regulation of YAP responsive elements revealed through large-scale functional epigenomic screens.

YAP is a key transcriptional co-activator of TEADs, it regulates cell growth and is frequently activated in cancer. In Malignant Pleural Mesothelioma (MPM), YAP is activated by loss-of-function mutations in upstream components of the Hippo pathway, while, in Uveal Melanoma (UM), YAP is activated in a Hippo-independent manner. To date, it is unclear if and how the different oncogenic lesions activating YAP impact its oncogenic program, which is particularly relevant for designing selective anti-cancer therapies. Here we show that, despite YAP being essential in both MPM and UM, its interaction with TEAD is unexpectedly dispensable in UM, limiting the applicability of TEAD inhibitors in this cancer type. Systematic functional interrogation of YAP regulatory elements in both cancer types reveals convergent regulation of broad oncogenic drivers in both MPM and UM, but also strikingly selective programs. Our work reveals unanticipated lineage-specific features of the YAP regulatory network that provide important insights to guide the design of tailored therapeutic strategies to inhibit YAP signaling across different cancer types

Inhibition of wild-type p53-expressing AML by novel small molecule HDM2 inhibitor, CGM097

The tumor suppressor, p53, is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (wt) p53, the Human Double Minute 2 (HDM2) protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell cycle arrest. We have characterized the novel HDM2 inhibitor, CGM097, as having significant activity against wt p53-expressing acute myeloid leukemia (AML). Specifically, CGM097 potently and selectively inhibited the proliferation of human AML cell lines and primary AML cells expressing wt p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against mutant FLT3-expressing cells, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. Taken together, these data suggest that CGM097 might be a promising treatment for AML characterized as harboring wt p53 as a single agent, as well as possibly in combination with another targeted therapy using tyrosine kinase inhibitors (TKIs) against oncogenes that drive AML

Large-scale functional epigenomic screens reveal cancer lineage-specific regulation of YAP responsive elements

YAP and TAZ are potent transcriptional co-factors engaging TEAD proteins downstream of Hippo signaling. Malignant pleural mesothelioma (MPM) and uveal melanoma (UM) are distinct cancer lineages bearing different genetic aberrations that ultimately lead to YAP activation. Here we use MPM and UM as prototypical cancers displaying, respectively, Hippo-dependent and -independent YAP activation to demonstrate that, while YAP is essential in both diseases, its interaction with TEAD is dispensable in UM, potentially limiting the application of TEAD inhibitors. Large scale functional epigenomic screens of YAP regulatory elements in MPM and UM reveal: 1) lineage-specific enhancers controlling broad oncogene dependencies (e.g., MYC) in both diseases, 2) rewiring of MAPK transcriptional regulatory networks in MPM, translating into synergistic efficacy of TEAD and MAPK inhibitors and 3) enrichment of melanocytic master regulators at functional YREs in UM. Our work prompts the design of tailored therapeutic strategies to inhibit YAP signaling in specific cancers

Complementary activities of DOT1L and Menin inhibitors in MLL-rearranged leukemia

Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.Leukemia advance online publication, 3 January 2017; doi:10.1038/leu.2016.327