Afatinib or Bevacizumab in combination with Osimertinib efficiently control tumor development in orthotopic murine models of non-small lung cancer.

Lung cancer is one of the most common and deadliest cancers. Preclinical models are essential to study new therapies and combinations taking tumor genetics into account. We have established cell lines expressing the luciferase gene from lines with varied genetic backgrounds, commonly encountered in patients with pulmonary adenocarcinoma. We have characterized these lines by testing their response to multiple drugs. Thus, we have developed orthotopic preclinical mouse models of NSCLC with very high engraftment efficiency. These models allow the easy monitoring of tumor growth, particularly in response to treatment, and of tumor cells dissemination in the body. We show that concomitant treatment with osimertinib (3rd generation tyrosine kinase inhibitor targeting mutated EGFR) and bevacizumab (anti-angiogenic targeting VEGF) can have a beneficial therapeutic effect on EGFR-mutated tumors. We also show that the addition of afatinib to osimertinib-treated tumors in escape leads to tumor growth inhibition. No such effect is observed with selumetinib or simvastatin. These preclinical mouse models therefore make it possible to test innovative therapeutic combinations and are also a tool of choice for studying resistance mechanisms.

KDM6B drives epigenetic reprogramming associated with lymphoid stromal cell early commitment and immune properties.

Mature lymphoid stromal cells (LSCs) are key organizers of immune responses within secondary lymphoid organs. Similarly, inflammation-driven tertiary lymphoid structures depend on immunofibroblasts producing lymphoid cytokines and chemokines. Recent studies have explored the origin and heterogeneity of LSC/immunofibroblasts, yet the molecular and epigenetic mechanisms involved in their commitment are still unknown. This study explored the transcriptomic and epigenetic reprogramming underlying LSC/immunofibroblast commitment. We identified the induction of lysine demethylase 6B (KDM6B) as the primary epigenetic driver of early immunofibroblast differentiation. In addition, we observed an enrichment for KDM6B gene signature in murine inflammatory fibroblasts and pathogenic stroma of patients with autoimmune diseases. Last, KDM6B was required for the acquisition of LSC/immunofibroblast functional properties, including the up-regulation of CCL2 and the resulting recruitment of monocytes. Overall, our results reveal epigenetic mechanisms that participate in the early commitment and immune properties of immunofibroblasts and support the use of epigenetic modifiers as fibroblast-targeting strategies in chronic inflammation.

UnAIDed Class Switching in Activated B-Cells Reveals Intrinsic Features of a Self-Cleaving IgH Locus.

Activation-induced deaminase (AID) is the major actor of immunoglobulin (Ig) gene diversification in germinal center B-cells. From its first description, it was considered as mandatory for class switch recombination (CSR), and this discovery initiated a long quest for all of the AID-interacting factors controlling its activity. The mechanisms focusing AID-mediated DNA lesions to given target sequences remain incompletely understood with regards the detailed characterization of optimal substrates in which cytidine deamination will lead to double strand breaks (DSBs) and chromosomal cleavage. In an effort to reconsider whether such CSR breaks absolutely require AID, we herein provide evidence, based on deep-sequencing approaches, showing that this dogma is not absolute in both human and mouse B lymphocytes. In activated B-cells from either AID-deficient mice or human AID-deficient patients, we report an intrinsic ability of the IgH locus to undergo "on-target" cleavage and subsequent synapsis of broken regions in conditions able to yield low-level CSR. DNA breaks occur in such conditions within the same repetitive S regions usually targeted by AID, but their repair follows a specific pathway with increased usage of microhomology-mediated repair. These data further demonstrate the role of AID machinery as not initiating de novo chromosomal cleavage but rather catalyzing a process which spontaneously initiates at low levels in an appropriately conformed IgH locus.

Fam72a enforces error-prone DNA repair during antibody diversification.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.

ING2 tumor suppressive protein translocates into mitochondria and is involved in cellular metabolism homeostasis.

ING2 (Inhibitor of Growth 2) is a tumor suppressor gene that has been implicated in critical biological functions (cell-cycle regulation, replicative senescence, DNA repair and DNA replication), most of which are recognized hallmarks of tumorigenesis occurring in the cell nucleus. As its close homolog ING1 has been recently observed in the mitochondrial compartment, we hypothesized that ING2 could also translocate into the mitochondria and be involved in new biological functions. In the present study, we demonstrate that ING2 is imported in the inner mitochondrial fraction in a redox-sensitive manner in human cells and that this mechanism is modulated by 14-3-3η protein expression. Remarkably, ING2 is necessary to maintain mitochondrial ultrastructure integrity without interfering with mitochondrial networks or polarization. We observed an interaction between ING2 and mtDNA under basal conditions. This interaction appears to be mediated by TFAM, a critical regulator of mtDNA integrity. The loss of mitochondrial ING2 does not impair mtDNA repair, replication or transcription but leads to a decrease in mitochondrial ROS production, suggesting a detrimental impact on OXPHOS activity. We finally show using multiple models that ING2 is involved in mitochondrial respiration and that its loss confers a protection against mitochondrial respiratory chain inhibition in vitro. Consequently, we propose a new tumor suppressor role for ING2 protein in the mitochondria as a metabolic shift gatekeeper during tumorigenesis.

Synthesis of peptidomimetics and chemo-biological tools for CD95/PLCγ1 interaction analysis.

The death receptor CD95 (also known as Fas) induces apoptosis through protein/protein association and the formation of the death-inducing signaling complex. On the other hand, in certain biological conditions, this receptor recruits different proteins and triggers the formation of another complex designated motility-inducing signaling complex, which promotes cell migration and inflammation. This pathway relies on a short sequence of CD95, called calcium-inducing domain (CID), which interacts with the phospholipase PLCγ1. To better understand how CID/PLCγ1 interaction occurs, we synthesized different α-AA peptides mimicking CID. Some of these peptidomimetics are as potent as the natural peptide to disrupt the CID/PLCγ1 interaction and cell migration, and showed improved pharmacokinetic properties. We also generated biotinyl- and palmitoyl-labelled peptidomimetics, useful chemico-biological tools to further explore the pro-inflammatory signal of CD95, which plays an important role in the pathogenesis of lupus and other autoimmune diseases.

Disrupting the CD95-PLCγ1 interaction prevents Th17-driven inflammation.

CD95L is a transmembrane ligand (m-CD95L) that is cleaved by metalloproteases to release a soluble ligand (s-CD95L). Unlike m-CD95L, interaction between s-CD95L and CD95 fails to recruit caspase-8 and FADD to trigger apoptosis and instead induces a Ca2+ response via docking of PLCγ1 to the calcium-inducing domain (CID) within CD95. This signaling pathway induces accumulation of inflammatory Th17 cells in damaged organs of lupus patients, thereby aggravating disease pathology. A large-scale screen revealed that the HIV protease inhibitor ritonavir is a potent disruptor of the CD95-PLCγ1 interaction. A structure-activity relationship approach highlighted that ritonavir is a peptidomimetic that shares structural characteristics with CID with respect to docking to PLCγ1. Thus, we synthesized CID peptidomimetics abrogating both the CD95-driven Ca2+ response and transmigration of Th17 cells. Injection of ritonavir and the CID peptidomimetic into lupus mice alleviated clinical symptoms, opening a new avenue for the generation of drugs for lupus patients.

Alterations of EDEM1 functions enhance ATF6 pro-survival signaling.

Activating transcription factor 6 alpha (referred to as ATF6 hereafter) is an endoplasmic reticulum (ER)-resident glycoprotein and one of the three sensors of the unfolded protein response (UPR). Upon ER stress, ATF6 is exported to the Golgi complex where it is cleaved by the S1P and S2P proteases thus releasing ATF6 cytosolic fragment and leading to the transcription of ATF6 target genes. In this study, we performed a phenotypic small-interfering RNA (siRNA) screening to better characterize the ER mechanisms involved in ATF6 activation upon ER stress. This revealed that silencing of ER-degradation-enhancing alpha-mannosidase-like protein-1 (EDEM1) increased the bioavailability of ER stress-induced ATF6 export to the Golgi complex through the stabilization of the natively unstable ATF6 protein. Moreover, we characterized a somatic variant of EDEM1 (N198I) found in hepatocellular carcinoma that alters ATF6 signaling and might provide a selective advantage to the transforming cells. Hence, our work confirms the natively unstable nature of ATF6 and links this property to potentially associated pro-oncogenic functions.

Dual IRE1 RNase functions dictate glioblastoma development.

Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1α (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1-dependent decay (RIDD) of RNA Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1-dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression.

The pVHL172 isoform is not a tumor suppressor and up-regulates a subset of pro-tumorigenic genes including TGFB1 and MMP13.

The von Hippel-Lindau (VHL) tumor suppressor gene is often deleted or mutated in ccRCC (clear cell renal cell carcinoma) producing a non-functional protein. The gene encodes two mRNA, and three protein isoforms (pVHL213, pVHL160 and pVHL172). The pVHL protein is part of an E3 ligase complex involved in the ubiquitination and proteasomal degradation of different proteins, particularly hypoxia inducible factors (HIF) that drive the transcription of genes involved in the regulation of cell proliferation, angiogenesis or extracellular matrix remodelling. Other non-canonical (HIF-independent) pVHL functions have been described. A recent work reported the expression of the uncharacterized protein isoform pVHL172 which is translated from the variant 2 by alternative splicing of the exon 2. This splice variant is sometimes enriched in the ccRCCs and the protein has been identified in the respective samples of ccRCCs and different renal cell lines. Functional studies on pVHL have only concerned the pVHL213 and pVHL160 isoforms, but no function was assigned to pVHL172. Here we show that pVHL172 stable expression in renal cancer cells does not regulate the level of HIF, exacerbates tumorigenicity when 786-O-pVHL172 cells were xenografted in mice. The pVHL172-induced tumors developed a sarcomatoid phenotype. Moreover, pVHL172 expression was shown to up regulate a subset of pro-tumorigenic genes including TGFB1, MMP1 and MMP13. In summary we identified that pVHL172 is not a tumor suppressor. Furthermore our findings suggest an antagonistic function of this pVHL isoform in the HIF-independent aggressiveness of renal tumors compared to pVHL213.

CD90 Expression Controls Migration and Predicts Dasatinib Response in Glioblastoma.

Purpose: CD90 (Thy-1) is a glycophosphatidylinositol-anchored glycoprotein considered as a surrogate marker for a variety of stem cells, including glioblastoma (GBM) stem cells (GSC). However, the molecular and cellular functions of CD90 remain unclear.Experimental Design: The function of CD90 in GBM was addressed using cellular models from immortalized and primary GBM lines, in vivo orthotopic mouse models, and GBM specimens' transcriptome associated with MRI features from GBM patients. CD90 expression was silenced in U251 and GBM primary cells and complemented in CD90-negative U87 cells.Results: We showed that CD90 is not only expressed on GSCs but also on more differentiated GBM cancer cells. In GBM patients, CD90 expression was associated with an adhesion/migration gene signature and with invasive tumor features. Modulation of CD90 expression in GBM cells dramatically affected their adhesion and migration properties. Moreover, orthotopic xenografts revealed that CD90 expression induced invasive phenotypes in vivo Indeed, CD90 expression led to enhanced SRC and FAK signaling in our GBM cellular models and GBM patients' specimens. Pharmacologic inhibition of these signaling nodes blunted adhesion and migration in CD90-positive cells. Remarkably, dasatinib blunted CD90-dependent GBM cell invasion in vivo and killed CD90high primary GSC lines.Conclusions: Our data demonstrate that CD90 is an actor of GBM invasiveness through SRC-dependent mechanisms and could be used as a predictive factor for dasatinib response in CD90high GBM patients. Clin Cancer Res; 23(23); 7360-74. ©2017 AACR.

CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice.

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.

Sunitinib combined with angiotensin-2 type-1 receptor antagonists induces more necrosis: a murine xenograft model of renal cell carcinoma.

BACKGROUND: Angiotensin-2 type-1 receptor antagonists not are only antihypertensive drugs but also can inhibit VEGF production. We hypothesised that adding telmisartan to sunitinib could potentiate the antiangiogenic effects. MATERIAL AND METHODS: 786-O cell lines were injected in nude mice. After tumor development, mice were divided into 4 groups: the first was the control group (DMSO), the second group was treated with sunitinib alone, the third group was treated with telmisartan alone, and the fourth group was treated with the combination. Drugs were orally administered every day for four weeks. Animals were sacrificed after treatment. Blood and tumor tissues were collected for analysis by immunohistochemistry, Western Blot, and ELISA methods. RESULTS: All animals developed a ccRCC and ten in each group were treated. Using a kinetic model, tumors tended to grow slower in the combination group compared to others (P = 0.06). Compared to sunitinib alone, the addition of telmisartan significantly increased tissue necrosis (P = 0.038). Central microvascular density decreased (P = 0.0038) as well as circulating VEGF (P = 0.003). There was no significant variation in proliferation or apoptosis markers. CONCLUSION: The combination of sunitinib and telmisartan revealed an enhancement of the blockage of the VEGF pathway on renal tumor resulting in a decrease in neoangiogenesis and an increase in necrosis.

Cytoplasmic PAR-3 protein expression is associated with adverse prognostic factors in clear cell renal cell carcinoma and independently impacts survival.

Clear cell renal cell carcinomas (ccRCCs) represent 70% of renal cancers, and several clinical and histolopathological factors are implicated in their prognosis. We recently demonstrated that the overexpression of PAR-3 protein encoded by the PARD3 gene could be implicated in renal oncogenesis. The object of this work was to study the association of intratumoral PAR-3 expression with known prognostic parameters and clinical outcome. In this aim, PAR-3 expression was assessed by immunohistochemistry in ccRCC tumors of 101 patients from 2003 to 2005. The immunostaining of PAR-3 was scored either as membranous (mPAR-3) or as both membranous and cytoplasmic (cPAR-3). Cytoplasmic PAR-3 was significantly associated with worse histopathological and clinical prognostic factors: Fuhrman grades 3 and 4, tumor necrosis, sarcomatoid component, adrenal invasion, renal and hilar fat invasion, eosinophilic component, a noninactivated VHL gene, higher tumor grade, lymph node involvement, metastasis, and worse clinical Eastern Cooperative Oncology Group and S classification scores. After multivariate analysis, 2 parameters were independently associated with cPAR-3: necrosis and eosinophilic components. In addition, cPAR-3 patients had shorter overall and progression-free survivals independently from strong prognostic validated factors like metastases. A cytoplasmic expression of PAR-3 is therefore implicated in worse clinical and pathological cancer features in ccRCC and could be useful to identify patients with high-risk tumors.

All anti-vascular endothelial growth factor drugs can induce 'pre-eclampsia-like syndrome': a RARe study.

BACKGROUND: Specific therapies that target vascular endothelial growth factor (VEGF) and its receptors have improved the survival of patients with metastatic cancers, but can induce side effects. Renal side effects (proteinuria, hypertension and renal failure) are underestimated. METHODS: The French RARe (Reins sous traitement Anti-VEGF Registre) study collects data on patients with cancer who had a renal biopsy because of major renal side effects during treatment with anti-VEGF drugs. RESULTS: We collected 22 renal biopsies performed 16.2±10.6 months after the beginning of treatment; of which 21 had hypertension, mean proteinuria was 2.97±2.00 g/day and mean serum creatinine, 134±117 µmol/L. Thrombotic microangiopathy (TMA) was observed in 21 biopsy specimens, sometimes associated with acute tubular necrosis (ATN; n=4). TMA histological lesions were more important than the biological signs of TMA could suggest. Patients with ATN of >20% had higher serum creatinine levels than those with only TMA (231 versus 95 µmol/L). Nephrin, podocin and synaptopodin were variably down-regulated in all renal biopsies. VEGF was down-regulated in all glomeruli. CONCLUSION: This study underlines the importance of regular clinical and biological cardiovascular and renal checking during all anti-VEGF therapies for cancer for early detection of renal dysfunction. Collaboration between oncologists and nephrologists is essential. In such cases, renal biopsy might help in appreciating the severity of the renal lesions and after multidisciplinary discussion whether or not it is safe to continue the treatment.

Identification of pVHL as a novel substrate for Aurora-A in clear cell renal cell carcinoma (ccRCC).

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of kidney cancer and is often characterized by mutations or deletions of the Von Hippel Lindau (VHL) tumour suppressor gene. Aurora gene family members are implicated in proper mitotic progression and spindle checkpoint function and play a crucial role in cancer progression. In the present study, we assessed the expression of Aurora-A in a cohort of 30 ccRCC with fully characterized VHL status (wt/wt or mut/del) and Fuhrman grade. Aurora-A transcript and protein levels were significantly increased in high Fuhrman grade tumours and in VHLwt/wt tumours. These results suggest that Aurora-A and VHL interact in the ccRCC. We demonstrated that the two proteins interact in vivo and identified the Ser72 on the sequence of VHL as the unique site phosphorylated by Aurora-A.

[Angiotensin-2 type 1 receptors (AT1R) and cancers]. / Récepteurs de type 1 à l'angiotensine-2 (AT1R) et cancers.

Recently, several meta-analysis suggested an increased risk of cancers linked to the use of antagonists of angiotensin-2 receptors or inhibitors of angiotensinogen converting enzyme. The results of epidemiological studies are conflicting. Meta-analysis as well as retrospective studies are not reliable and biased, since they have never been designed to explore any pro- or antitumoral effect. We lack of prospective studies that could take off the doubt on these drugs. Nevertheless, all experimental researches pointed out potent antitumoral properties. Indeed, direct antiproliferative and neo-angiogenic inhibition have been described on tumor cell cultures as well as on animal models. Moreover, we are convinced that the use of antagonists of angiotensin-2 receptors and inhibitors of angiotensinogen converting enzyme may be then of clinical use in the near future in association with classical antitumor drugs. In this review, we proposed to explore these data by a thorough analysis of recent literature associating epidemiological and experimental studies.

Description of 2 angiogenic phenotypes in clear cell renal cell carcinoma.

Angiogenesis in clear cell renal cell carcinoma has received recent focus with the development of antiangiogenic therapies. Although tumor progression is known to be correlated with intratumoral and plasma levels of vascular endothelial growth factor-A, the role of tumor induced-angiogenesis remains unclear in these tumors. We analyzed the vascular network in a cohort of 73 clear cell renal cell carcinoma cases using endothelial immunostaining. We studied protein expression of vascular endothelial growth factor, Von Hippel Lindau, and carbonic anhydrase IX by immunohistochemistry, Von Hippel Lindau gene alteration by sequencing, deletion- and methylation-specific Multiplex Ligation-dependent Probe Amplification, and gene expression by pangenomic microarray and quantitative polymerase chain reaction in a subcohort of 39 clear cell renal cell carcinoma cases. We described 2 distinct angiogenic phenotypes in comparison with the normal kidney vasculature: low and high angiogenic phenotypes. The low angiogenic phenotype was associated with more aggressive prognostic factors such as T3 to T4 (62% versus 31%, P=.002), N+ (29% versus 3% P=.004), M+ (53% versus 21%, P=.004) stages, Fuhrman grade (grade 3-4: 91% versus 36%, P<.001), and intratumoral vascular endothelial growth factor expression (74% versus 28%, P<.001); was less associated with Von Hippel Lindau inactivation (56% versus 80%, P=.03); and was a predictor of poor prognosis in terms of progression-free, cancer-specific, and overall survival (log-rank test, P=.002, P=.011, and P=.035, respectively). The low angiogenic phenotype was also associated with a relative down-regulation of gene expression (platelet-derived growth factor D, N-acetyl transferase 8, and N-acetyl transferase 8 B). In conclusion, the histologic and molecular distinction between these 2 angiogenic phenotypes could help to better understand the biologic behavior of clear cell renal cell carcinoma angiogenesis and could be analyzed in a prospective study of the effects of antiangiogenic drugs.