EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread.

In the present study, we provide a comparative phenotypic and genotypic analysis of azacitidine-sensitive and resistant SKM-1 cell lines. Morphologically, SKM1-R exhibited increase in cell size that accounts for by enhanced ploidy in a majority of cells as shown by cell cycle and karyotype analysis. No specific Single Nucleotide Polymorphism (SNP) alteration was found in SKM1-R cells compared to their SKM1-S counterpart. Comparative pangenomic profiling revealed the up-regulation of a panel of genes involved in cellular movement, cell death and survival and down-regulation of genes required for cell to cell signaling and free radical scavenging in SKM1-R cells. We also searched for mutations frequently associated with myelodysplastic syndromes (MDS) and found that both cell lines harbored mutations in TET2, ASLX1 and TP53. Collectively, our data show that despite their different morphological and phenotypic features, SKM1-S and SKM1-R cells exhibited similar genotypic characteristics. Finally, pangenomic profiling identifies new potential pathways to be targeted to circumvent AZA-resistance. In conclusion, SKM1-R cells represent a valuable tool for the validation of new therapeutic intervention in MDS.

A new C47 polyoxygenated acetylenic acid, nepheliosyne B (2), along with the previously described nepheliosyne A (1), have been isolated from the New Caledonian marine sponge Niphates sp. Their structures have been elucidated on the basis of extensive spectroscopic analyses. These metabolites exhibited a moderate cytotoxicity against K562, U266, SKM1, and Kasumi cancer cell lines.

Simalikalactone E (SkE) is a quassinoid extracted from a widely used Amazonian antimalarial remedy. Although SkE has previously been shown to have cytostatic and/or cytotoxic activities in some tumor cell lines, its mechanism of action has not yet been characterized. We show here that SkE in the high nanomolar range inhibited the growth of various leukemic and solid tumor cell lines. Importantly, SkE was highly efficient at inhibiting chronic myelogenous leukemia (CML) cells that exhibit constitutive activation of the MAPK pathway and, accordingly, it impaired the phosphorylation of ERK1/2. SkE also abrogated MEK1/2 and B-Raf phosphorylation but had no effect on Ras activity. Moreover, SkE was particularly effective against melanoma cell lines carrying the B-Raf-V600E mutation. Importantly, SkE resensitized the PLX-4032-resistant 451Lu melanoma cell line (451Lu-R) and was more efficient than U0126, a MEK inhibitor, and PLX-4032 (PLX) at inducing the apoptosis of two hairy cell leukemia (HCL) patient samples carrying the B-Raf-V600E mutation. Finally, SkE was as efficient as imatinib at inhibiting tumor formation in a xenograft model of CML cells in athymic mice. In conclusion, we show that SkE, a very potent inhibitor of B-Raf-V600E, is highly effective against cancer cell lines that exhibit constitutive activation of the ERK1/2 pathway.

Dysregulated immune response is the key factor leading to unfavorable coronavirus disease 2019 (COVID-19) outcome. Depending on the pathogen-associated molecular pattern, the NLRP3 inflammasome can play a crucial role during innate immunity activation. To date, studies describing the NLRP3 response during severe acute respiratory syndrome coronavirus 2 infection in patients are lacking. We prospectively monitored caspase-1 activation levels in peripheral myeloid cells from healthy donors and patients with mild to critical COVID-19. The caspase-1 activation potential in response to NLRP3 inflammasome stimulation was opposed between nonclassical monocytes and CD66b+CD16dim granulocytes in severe and critical COVID-19 patients. Unexpectedly, the CD66b+CD16dim granulocytes had decreased nigericin-triggered caspase-1 activation potential associated with an increased percentage of NLRP3 inflammasome impaired immature neutrophils and a loss of eosinophils in the blood. In patients who recovered from COVID-19, nigericin-triggered caspase-1 activation potential in CD66b+CD16dim cells was restored and the proportion of immature neutrophils was similar to control. Here, we reveal that NLRP3 inflammasome activation potential differs among myeloid cells and could be used as a biomarker of a COVID-19 patient's evolution. This assay could be a useful tool to predict patient outcome. This trial was registered at www.clinicaltrials.gov as #NCT04385017.

Polo-like kinase 1 (Plk1) expression is inversely correlated with survival advantages in many cancers. However, molecular mechanisms that underlie Plk1 expression are poorly understood. Here, we uncover a hypoxia-regulated mechanism of Plk1-mediated cancer metastasis and drug resistance. We demonstrated that a HIF-2-dependent regulatory pathway drives Plk1 expression in clear cell renal cell carcinoma (ccRCC). Mechanistically, HIF-2 transcriptionally targets the hypoxia response element of the Plk1 promoter. In ccRCC patients, high expression of Plk1 was correlated to poor disease-free survival and overall survival. Loss-of-function of Plk1 in vivo markedly attenuated ccRCC growth and metastasis. High Plk1 expression conferred a resistant phenotype of ccRCC to targeted therapeutics such as sunitinib, in vitro, in vivo, and in metastatic ccRCC patients. Importantly, high Plk1 expression was defined in a subpopulation of ccRCC patients that are refractory to current therapies. Hence, we propose a therapeutic paradigm for improving outcomes of ccRCC patients.

PIK3CA, which encodes the p110α catalytic subunit of PI3Kα, is one of the most frequently altered oncogenes in colon cancer (CC), but its prognostic value is still a matter of debate. Few reports have addressed the association between PIK3CA mutations and survival and their results are controversial. In the present study, we aimed to clarify the prognostic impact of PIK3CA mutations in stage I-III CC according to mismatch repair status. Fresh frozen tissue samples from two independent cohorts with a total of 826 patients who underwent curative surgical resection of CC were analyzed for microsatellite instability and screened for activating point mutations in exon 9 and 20 of PIK3CA by direct sequencing. Overall, 693 tumors (84%) exhibited microsatellite stability (MSS) and 113 samples (14%) harbored PIK3CA mutation. In the retrospective training cohort (n = 433), patients with PIK3CA-mutated MSS tumors (n = 47) experienced a significant increased 5-year relapse-free interval compared with PIK3CA wild-type MSS tumors (n = 319) in univariate analysis (94% vs. 68%, Log-rank P = 0. 0003) and in multivariate analysis (HR = 0.12; 95% confidence interval, 0.029-0.48; P = 0.0027). In the prospective validation cohort (n = 393), the favorable prognostic impact of PIK3CA mutations in MSS tumors (n = 327) was confirmed (83% vs. 67%, Log-rank P = 0.04). Our study showed that PIK3CA mutations are associated with a good prognosis in patients with MSS stage I-III CC.

We showed previously that Lyn is a substrate for caspases, a family of cysteine proteases, involved in the regulation of apoptosis and inflammation. Here, we report that expression of the caspase-cleaved form of Lyn (LynDeltaN), in mice, mediates a chronic inflammatory syndrome resembling human psoriasis. Genetic ablation of TNF receptor 1 in a LynDeltaN background rescues a normal phenotype, indicating that LynDeltaN mice phenotype is TNF-alpha-dependent. The predominant role of T cells in the disease occurring in LynDeltaN mice was highlighted by the distinct improvement of LynDeltaN mice phenotype in a Rag1-deficient background. Using pan-genomic profiling, we also established that LynDeltaN mice show an increased expression of STAT-3 and inhibitory members of the NFkappaB pathway. Accordingly, LynDeltaN alters NFkappaB activity underlying a link between inhibition of NFkappaB and LynDeltaN mice phenotype. Finally, analysis of Lyn expression in human skin biopsies of psoriatic patients led to the detection of Lyn cleavage product whose expression correlates with the activation of caspase 1. Our data identify a new role for Lyn as a regulator of psoriasis through its cleavage by caspases.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

Azacitidine is the leading compound to treat patients suffering myelodysplastic syndrome (MDS) or AML with less than 30% of blasts, but a majority of patients is primary refractory or rapidly relapses under treatment. These patients have a drastically reduced life expectancy as compared to sensitive patients. Therefore identifying predictive factors for AZA resistance is of great interest to propose alternative therapeutic strategies for non-responsive patients. We generated AZA-resistant myeloid cell line (SKM1-R) that exhibited increased expression of BCL2L10 an anti-apoptotic Bcl-2 member. Importantly, BCL2L10 knockdown sensitized SKM1-R cells to AZA effect suggesting that increased BCL2L10 expression is linked to AZA resistance in SKM1-R. We next established in 77 MDS patients that resistance to AZA is significantly correlated with the percentage of MDS or AML cells expressing BCL2L10. In addition, we showed that the proportion of BCL2L10 positive bone marrow cells can predict overall survival in MDS or AML patients. We propose a convenient assay in which the percentage of BCL2L10 expressing cells as assessed by flow cytometry is predictive of whether or not a patient will become resistant to AZA. Therefore, systematic determination of BCL2L10 expression could be of great interest in newly diagnosed and AZA-treated MDS patients.

AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

Understanding the functional role of mutated genes in cancer is required to translate the findings of cancer genomics into therapeutic improvement. BTG1 is recurrently mutated in the MCD/C5 subtype of diffuse large B-cell lymphoma (DLBCL), which is associated with extranodal dissemination. Here, we provide evidence that Btg1 knock out accelerates the development of a lethal lymphoproliferative disease driven by Bcl2 overexpression. Furthermore, we show that the scaffolding protein BCAR1 is a BTG1 partner. Moreover, after BTG1 deletion or expression of BTG1 mutations observed in patients with DLBCL, the overactivation of the BCAR1-RAC1 pathway confers increased migration ability in vitro and in vivo. These modifications are targetable with the SRC inhibitor dasatinib, which opens novel therapeutic opportunities in BTG1 mutated DLBCL.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR -mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR -mutant NSCLC cells.

Abiraterone acetate (AA) is the first-in-class of drugs belonging to the second-generation of agents inhibiting androgen neosynthesis in advanced prostate cancer. A cumulative experience attests that germinal gene polymorphisms may play a role in the prediction of anticancer agent pharmacodynamics variability. In the present prospective, multicentric study, gene polymorphisms of CYP17A1 (AA direct target) and the androgen transporter genes SLCO2B1 and SLCO1B3 (potential modulators of AA activity) were confronted with AA pharmacodynamics (treatment response and toxicity) in a group of 137 advanced prostate cancer patients treated in the first line by AA. The median follow-up was 56.3 months (95% CI [52.5-61]). From multivariate analysis, rs2486758 C/C (CYP17A1) and PSA (≥10 ng/mL) were associated with a shorter 3-year biological PFS (HR = 4.05, IC95% [1.46-11.22]; p = 0.007 and HR = 2.08, IC95% [1.31-3.30]; p = 0.002, respectively). From a multivariate analysis, the rs743572 (CYP17A1) and performance status were independently associated with significant toxicity (OR = 3.78 (IC95% [1.42-9.75]; p = 0.006 and OR = 4.54; IC95% [1.46-13.61]; p = 0.007, respectively). Host genome characteristics may help to predict AA treatment efficacy and identify patients at risk for toxicity.

CSF-1 and IL-34 share the CSF-1 receptor and no differences have been reported in the signaling pathways triggered by both ligands in human monocytes. IL-34 promotes the differentiation and survival of monocytes, macrophages and osteoclasts, as CSF-1 does. However, IL-34 binds other receptors, suggesting that differences exist in the effect of both cytokines. In the present study, we compared the differentiation and polarization abilities of human primary monocytes in response to CSF-1 or IL-34. CSF-1R engagement by one or the other ligands leads to AKT and caspase activation and autophagy induction through expression and activation of AMPK and ULK1. As no differences were detected on monocyte differentiation, we investigated the effect of CSF-1 and IL-34 on macrophage polarization into the M1 or M2 phenotype. We highlighted a striking increase in IL-10 and CCL17 secretion in M1 and M2 macrophages derived from IL-34 stimulated monocytes, respectively, compared to CSF-1 stimulated monocytes. Variations in the secretome induced by CSF-1 or IL-34 may account for their different ability to polarize naïve T cells into Th1 cells. In conclusion, our findings indicate that CSF-1 and IL-34 exhibit the same ability to induce human monocyte differentiation but may have a different ability to polarize macrophages.

Differentiation-inducing factor (DIF) defines a group of chlorinated hexaphenones that orchestrate stalk-cell differentiation in the slime mold Dictyostelium discoideum (DD). DIF-1 and 3 have also been reported to have tumor inhibiting properties; however, the mechanisms that underlie the effects of these compounds remain poorly defined. Herein, we show that DIF-3 rapidly triggers Ca2+ release and a loss of mitochondrial membrane potential (MMP) in the absence of cytochrome c and Smac release and without caspase activation. Consistently with these findings, we also detected no evidence of apoptosis in cells treated with DIF-3 but instead found that this compound induced autophagy. In addition, DIF-3 promoted mitochondrial fission in K562 and HeLa cells, as assessed by electron and confocal microscopy analysis. Importantly, DIF-3 mediated the phosphorylation and redistribution of dynamin-related protein 1 (DRP1) from the cytoplasmic to the microsomal fraction of K562 cells. Pharmacological inhibition or siRNA silencing of DRP1 not only inhibited mitochondrial fission but also protected K562 cells from DIF-3-mediated cell death. Furthermore, DIF-3 potently inhibited the growth of imatinib-sensitive and imatinib-resistant K562 cells. It also inhibited tumor formation in athymic mice engrafted with an imatinib-resistant CML cell line. Finally, DIF-3 exhibited a clear selectivity toward CD34+ leukemic cells from CML patients, compared with CD34- cells. In conclusion, we show that the potent anti-leukemic effect of DIF-3 is mediated through the induction of mitochondrial fission and caspase-independent cell death. Our findings may have important therapeutic implications, especially in the treatment of tumors that exhibit defects in apoptosis regulation.

The advent of tyrosine kinase inhibitor (TKI) therapy has considerably improved the survival of patients suffering chronic myelogenous leukemia (CML). Indeed, inhibition of BCR-ABL by imatinib, dasatinib or nilotinib triggers durable responses in most patients suffering from this disease. Moreover, resistance to imatinib due to kinase domain mutations can be generally circumvented using dasatinib or nilotinib, but the multi-resistant T315I mutation that is insensitive to these TKIs, remains to date a major clinical problem. In this line, ponatinib (AP24534) has emerged as a promising therapeutic option in patients with all kinds of BCR-ABL mutations, especially the T315I one. However and surprisingly, the effect of ponatinib has not been extensively studied on imatinib-resistant CML cell lines. Therefore, in the present study, we used several CML cell lines with different mechanisms of resistance to TKI to evaluate the effect of ponatinib on cell viability, apoptosis and signaling. Our results show that ponatinib is highly effective on both sensitive and resistant CML cell lines, whatever the mode of resistance and also on BaF3 murine B cells carrying native BCR-ABL or T315I mutation. We conclude that ponatinib could be effectively used for all types of TKI-resistant patients.

Colon cancer (CC) pathological staging fails to accurately predict recurrence, and to date, no gene expression signature has proven reliable for prognosis stratification in clinical practice, perhaps because CC is a heterogeneous disease. The aim of this study was to establish a comprehensive molecular classification of CC based on mRNA expression profile analyses.

Transgenic mice expressing the caspase-cleaved form of the tyrosine kinase Lyn (LynΔN) develop a TNFα-dependent skin disease that accurately recapitulates human psoriasis. Participation of lymphocytes in this disease was confirmed by backcrossing LynΔN mice on a Rag-1 deficient background. The present study was therefore conducted to analyze whether modification of lymphocyte homeostasis does occur and participate in the phenotype of LynΔN mice. We show here that LynΔN mice consistently exhibit thymic atrophy that correlates with both a net decrease in the CD4+/CD8+ Double Positive (DP) and an increase in Single Positive (SP) thymocyte sub-populations, but also display an increase of splenic mature B cell. Interestingly, a normal immune phenotype was rescued in a TNFR1 deficient background. Finally, none of these immune alterations was detected in newborn mice before the onset of inflammation. Therefore, we conclude that chronic inflammation can induce thymic atrophy and perturb spleen homeostasis in LynΔN mice through the increased production of TNFα, LTß and TNFR1 signaling.

Deficiency in dihydropyrimidine dehydrogenase (DPD) enzyme is the main cause of severe and lethal fluoropyrimidine-related toxicity. Various approaches have been developed for DPD-deficiency screening, including DPYD genotyping and phenotyping. The goal of this prospective observational study was to perform exhaustive exome DPYD sequencing and to examine relationships between DPYD variants and toxicity in advanced breast cancer patients receiving capecitabine.