NCI-MATCH Arms N & P: Phase II study of PI3K beta inhibitor GSK2636771 in patients (pts) with cancers (ca) with PTEN mutation/deletion (mut/del) or PTEN protein loss

Background: The NCI-MATCH trial is the largest national study (1173 sites) for ptswith relapsed/ refractory solid tumors, lymphomas and myeloma, which assigns tar-geted therapies based on individual tumor molecular alterations detected using theadapted Oncomine AmpliSeq panel (143 genes) and immunohistochemistry (IHC).We hypothesized that patients with PTEN-deficient cancers enrolled to Arms N and Pmay benefit from treatment with the PI3K beta-selective inhibitor GSK2636771. Methods: Eligibility: relapsed/refractory ca, good end-organ function, and ECOG PS ≤ 1. Pts were screened for molecular alterations by centralized testing on fresh tumor biopsy and had deleterious PTEN mut/del without loss of expression (Arm N) or complete loss of cytoplasmic and nuclear PTEN staining on IHC (Arm P), and no other aberrations activating the PI3K/MTOR and MAPK pathways (mut in PIK3CA, PIK3R1, BRAF, KRAS, AKT1, TSC1/2, mTOR, RHEB, NF2, NRAS, HRAS). Pts received GSK2636771 400mg/day (28-days cycles). RECIST 1.1 overall response rate (ORR) was the primary endpoint. Results: Of 59 enrolled pts, 56 were eligible and received treatment. Of 22 pts with PTEN mut/del (Arm N: 6 uterine, 2 breast, 2 prostate, 2 head/neck ca, 10 other), all are off treatment as of analysis (14 disease progression, 4 for adverse events [AEs], 4 other). One pt (4.5%) with prostate ca (PTEN deletion, MPRSS2-ERG fusion) attained a partial response (-42%). Of 7 (32%) pts with stable disease (SD), 2 had SD \u3e 6 months (uterine leiomyosarcoma; endometrial carcinoma). Of 34 pts with loss of PTEN protein by IHC (Arm P: 7 prostate, 6 breast, 3 squamous anal ca, 2 cholangiocarcinoma, 16 other), all are off treatment as of analysis (26 disease progression, 4 for AE, 4 other). Of 9 (37.5%) pts with SD, 3 had SD \u3e 6 months (prostate cancer; squamous bladder cancer, squamous anal cancer). Median progression-free survival was 1.8 months for both arms. Gr ≥ 3 treatment-related (tr) reversible toxicities were experienced by 30% (7) and 20% (7) of pts in arms N and P, respectively. No tr Gr 5 toxicities were observed in either arm. Conclusions: Single agent GSK2636771 has very modest activity in ca with PTEN gene mutation/deletion and/or PTEN protein loss

BRAF V600E mutations in urine and plasma cell-free DNA from patients with Erdheim-Chester disease.

Erdheim-Chester disease (ECD) is a rare histiocytosis with a high prevalence of BRAF V600E mutation (>50% of patients). Patients with BRAF-mutant ECD can respond to BRAF inhibitors. Unfortunately, the lack of adequate archival tissue often precludes BRAF testing. We hypothesized that cell-free DNA (cfDNA) from plasma or urine can offer an alternative source of biologic material for testing. We tested for BRAF V600E mutation in cfDNA from the plasma and urine of 6 ECD patients. In patients with available archival tissue, the result of BRAF mutation analysis was concordant with plasma and urine cfDNA results in all 3 patients (100% agreement, kappa 1.00). In all 6 patients, BRAF mutation analysis of plasma and urine cfDNA was concordant in 5 of 6 patients (83% agreement, kappa 0.67). Testing for BRAF V600E mutation in plasma and urine cfDNA should be further investigated as an alternative to archival tissue mutation analysis

WNT signalling in prostate cancer

Genome sequencing and gene expression analyses of prostate tumours have highlighted the potential importance of genetic and epigenetic changes observed in WNT signalling pathway components in prostate tumours-particularly in the development of castration-resistant prostate cancer. WNT signalling is also important in the prostate tumour microenvironment, in which WNT proteins secreted by the tumour stroma promote resistance to therapy, and in prostate cancer stem or progenitor cells, in which WNT-β-catenin signals promote self-renewal or expansion. Preclinical studies have demonstrated the potential of inhibitors that target WNT receptor complexes at the cell membrane or that block the interaction of β-catenin with lymphoid enhancer-binding factor 1 and the androgen receptor, in preventing prostate cancer progression. Some WNT signalling inhibitors are in phase I trials, but they have yet to be tested in patients with prostate cancer

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.FEDER through POFC – COMPETE and by Fundação para a Ciência e Tecnologia through projects PEst-OE/BIA/UI4050/2014 and FCT ANR/BEX-BCM/0175/201

Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer

The significance of heat transport by shallow fluid flow at an active plate boundary: the Southern Alps, New Zealand

Fluid flow can influence fault behavior. Here we quantify the role of groundwater heat advection in establishing the thermal structure of the Alpine Fault, a major tectonic boundary in southern New Zealand that accommodates most of the motion between the Australian and Pacific Plates. Convergence on the Alpine Fault has rapidly uplifted the Southern Alps, resulting in high geothermal gradients and a thin seismogenic zone. A new equilibrium temperature profile from the 818-m-deep Deep Fault Drilling Project 2B borehole has been interrogated using one-dimensional analytical models of fluid and rock advection. Models indicate a total heat flux of 720-mW m2 results from groundwater flow with Darcy velocities approximating to 7.8 × 1010 m s1. Groundwaters advect significantly more heat than rock advection in the shallow orogen (<6-km depth) and are the major control on the subsurface temperature field

PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma

Background Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. Methods We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. Results Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. Conclusions Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer

Grb2 depletion under non-stimulated conditions inhibits PTEN, promotes Akt-induced tumor formation and contributes to poor prognosis in ovarian cancer

In the absence of extracellular stimulation the adaptor protein growth factor receptor-bound protein (Grb2) and the phospholipase Plcγ1 compete for the same binding site on fibroblast growth factor receptor 2 (FGFR2). Reducing cellular Grb2 results in upregulation of Plcγ1 and depletion of the phospholipid PI(4,5)P2. The functional consequences of this event on signaling pathways are unknown. We show that the decrease in PI(4,5)P2 level under non-stimulated conditions inhibits PTEN activity leading to the aberrant activation of the oncoprotein Akt. This results in excessive cell proliferation and tumor progression in a xenograft mouse model. As well as defining a novel mechanism of Akt phosphorylation with important therapeutic consequences, we also demonstrate that differential expression levels of FGFR2, Plcγ1 and Grb2 correlate with patient survival. Oncogenesis through fluctuation in the expression levels of these proteins negates extracellular stimulation or mutation and defines them as novel prognostic markers in ovarian cancer

Up-regulated expression of LAMP2 and autophagy activity during neuroendocrine differentiation of prostate cancer LNCaP cells

Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and βIII tubulin (βIII tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1μM AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer

Pemetrexed Induced Thymidylate Synthase Inhibition in Non-Small Cell Lung Cancer Patients: A Pilot Study with 3 '-Deoxy-3 '-[F-18]fluorothymidine Positron Emission Tomography

OBJECTIVES: Pemetrexed is a thymidylate synthase (TS) inhibitor and is effective in non-small cell lung cancer (NSCLC). 3'-deoxy-3'-[¹⁸F]fluorothymidine (¹⁸F-FLT), a proliferation marker, could potentially identify tumor specific TS-inhibition. The aim of this study was to investigate the effect of pemetrexed-induced TS-inhibition on ¹⁸F-FLT uptake 4 hours after pemetrexed administration in metastatic NSCLC patients. METHODS: Fourteen NSCLC patients underwent dynamic ¹⁸F-FLT positron emission tomography (PET) scans at baseline and 4 hours after the first dose of pemetrexed. Volumes of interest were defined with a 41%, 50% and 70% threshold of the maximum pixel. Kinetic analysis and simplified measures were performed. At one, two, four and six hours after pemetrexed, plasma deoxyuridine was measured as systemic indicator of TS-inhibition. Tumor response measured with response evaluation criteria in solid tumors (RECIST), time to progression (TTP) and overall survival (OS) were determined. RESULTS: Eleven patients had evaluable ¹⁸F-FLT PET scans at baseline and 4 hours after pemetrexed. Two patients had increased ¹⁸F-FLT uptake of 35% and 31% after pemetrexed, whereas two other patients had decreased uptake of 31%. In the remaining seven patients ¹⁸F-FLT uptake did not change beyond test-retest borders. In all patients deoxyuridine levels raised after administration of pemetrexed, implicating pemetrexed-induced TS-inhibition. ¹⁸F-FLT uptake in bone marrow was significantly increased 4 hours after pemetrexed administration. Six weeks after the start of treatment 5 patients had partial response, 4 stable disease and 2 progressive disease. Median TTP was 4.2 months (range 3.0-7.4 months); median OS was 13.0 months (range 5.1-30.8 months). Changes in ¹⁸F-FLT uptake were not predictive for tumor response, TTP or OS. CONCLUSIONS: Measuring TS-inhibition in a clinical setting 4 hours after pemetrexed revealed a non-systematic change in ¹⁸F-FLT uptake within the tumor. No significant association with tumor response, TTP or OS was observed