Abstract only Background: Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule that regulates signalling cascades and gene transcription. We have uncovered vasculoprotective and ...atheroprotective effects of FHL2 knockout in mice. Since B cells could regulate these processes, we investigated the potential role of FHL2 in B cell function and activity. Methods and Results: Under basal conditions, FHL2-/- mice presented a mild splenomegaly (84.0±9.0 vs 68.0±5.0mg), associated with bigger spleen follicles (1.6 fold) and higher proportions of spleen B cells (56.0±2.0 vs 40.0±9.0%) vs WT mice. Flow cytometry confirmed significantly higher (p<0.05) proportions of B cells in the follicles (Fo)(CD23+CD21lo/int) (77.0±1.0 vs 67.0±3.0%) and the marginal zone (Mz)(CD23-,CD21+)(7.0±1.0 vs 2.5±1.0%) of FHL2-/- vs WT mice. Mice were injected with sheep red blood cells (SRBC) to elicit a T cell-dependent B cell activation. SRBC did not influence Fo and Mz B cell numbers, but it did affect germinal center (GC) formation. GCs are zones within Fo where T cells activate B cells to produce high affinity antibodies. SRBC induced a 5- and 2.5-fold increase in B cells (GL7+) within FHL2-/- and WT GCs, respectively. However, there was no increase in plasma IgG1 levels in FHL2-/- mice in response to SRBC, whereas a 3-fold increase was observed in WT mice (1158±227 vs 2943±226ng/ml; p<0.05). Instead, plasma IgM levels were higher in FHL2-/- than WT SRBC mice. Moreover, SRBC only induced a modest, 1.3-fold fold increase in Fo T helper cells (CXCR5+PD1+) in FHL2-/- compared with a 6-fold increase in WT. Nevertheless, FHL2-/- B cells successfully proliferated and underwent class switch recombination in response to LPS, anti-CD40 and IL-4 in vitro, indicating that these cells were not defective. Conclusion: These results suggest that FHL2 plays a critical role in spleen antibody production in response to a T-dependent antigen, possibly by affecting activation or antigen presentation by Fo T helper cells.
Promoting the macroautophagy/autophagy-mediated degradation of specific proteins and organelles can potentially be utilized to induce apoptosis in cancer cells or sensitize tumor cells to therapy. To ...examine this concept, we enriched for autophagosomes from histone deacetylase inhibitor (HDACi)-sensitive U937 lymphoma cells and isogenic HDACi-resistant cells. Mass spectrometry on autophagosome-enriched fractions revealed that HDACi-resistant cells undergo elevated pexophagy, or autophagy of the peroxisome, an organelle that supports tumor growth. To disturb peroxisome homeostasis, we enhanced pexophagy in HDACi-resistant cells via genetic silencing of peroxisome exportomer complex components (PEX1, PEX6, or PEX26). This consequently sensitized resistant cells to HDACi-mediated apoptosis, which was rescued by inhibiting ATM/ataxia-telangiectasia mutated (ATM serine/threonine kinase), a mediator of pexophagy. We subsequently engineered melanoma cells to stably repress PEX26 using CRISPR interference (CRISPRi). Melanoma cells with repressed PEX26 expression showed evidence of both increased pexophagy and peroxisomal matrix protein import defects versus single guide scrambled (sgSCR) controls. In vivo studies showed that sgPEX26 melanoma xenografts recurred less compared to sgSCR xenografts, following the development of resistance to mitogen-activated protein kinase (MAPK)-targeted therapy. Finally, prognostic analysis of publicly available datasets showed that low expression levels of PEX26, PEX6 and MTOR, were significantly associated with prolonged patient survival in lymphoma, lung cancer and melanoma cohorts. Our work highlighted that drugs designed to disrupt peroxisome homeostasis may serve as unconventional therapies to combat therapy resistance in cancer.
Abbreviations: ABCD3/PMP70: ATP binding cassette subfamily D member 3; ACOX1: acyl-CoA oxidase 1; AP: autophagosome; COX: cytochrome c oxidase; CQ: chloroquine; CRISPRi: clustered regularly interspaced short palindromic repeats interference; DLBCL: diffuse large B-cell lymphoma; GO: gene ontology; dCas9: Cas9 endonuclease dead, or dead Cas9; HDACi: histone deacetylase inhibitors; IHC: Immunohistochemistry; LAMP2: lysosomal associated membrane protein 2; LCFAs: long-chain fatty acids; LFQ-MS: label-free quantitation mass spectrometry; LPC: lysophoshatidylcholine; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PBD: peroxisome biogenesis disorders; PTS1: peroxisomal targeting signal 1; ROS: reactive oxygen species; sgRNA: single guide RNA; VLCFAs: very-long chain fatty acids; Vor: vorinostat; WO: wash-off.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Several immune regulatory cell types participate in the protection against autoimmune diseases such as autoimmune diabetes. Of these immunoregulatory cells, we and others have shown that peripheral ...CD4
−
CD8
−
double negative (DN) T cells can induce antigen-specific immune tolerance. Particularly, we have described that diabetes-prone mice exhibit a lower number of peripheral DN T cells compared to diabetes-resistant mice. Identifying the molecular pathways that influence the size of the DN T cell pool in peripheral lymphoid organs may thus be of interest for maintaining antigen-specific immune tolerance. Hence, through immunogenetic approaches, we found that two genetic loci linked to autoimmune diabetes susceptibility, namely
Idd2
and
Idd13
, independently contribute to the partial restoration of DN T cell proportion in secondary lymphoid organs. We now extend these findings to show an interaction between the
Idd2
and
Idd13
loci in determining the number of DN T cells in secondary lymphoid organs. Using bioinformatics tools, we link potential biological pathways arising from interactions of genes encoded within the two loci. By focusing on cell cycle, we validate that both the
Idd2
and
Idd13
loci influence RAD51 expression as well as DN T cell progression through the cell cycle. Altogether, we find that genetic interactions between
Idd2
and
Idd13
loci modulate cell cycle progression, which contributes, at least in part, to defining the proportion of DN T cells in secondary lymphoid organs.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
•Mitochondrial pyruvate import plays a critical role in regulating bioenergetic and proteolytic capacities of MM cells.
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Multiple myeloma (MM) is a hematological malignancy that ...emerges from antibody-producing plasma B cells. Proteasome inhibitors, including the US Food and Drug Administration–approved bortezomib (BTZ) and carfilzomib (CFZ), are frequently used for the treatment of patients with MM. Nevertheless, a significant proportion of patients with MM are refractory or develop resistance to this class of inhibitors, which represents a significant challenge in the clinic. Thus, identifying factors that determine the potency of proteasome inhibitors in MM is of paramount importance to bolster their efficacy in the clinic. Using genome-wide CRISPR-based screening, we identified a subunit of the mitochondrial pyruvate carrier (MPC) complex, MPC1, as a common modulator of BTZ response in 2 distinct human MM cell lines in vitro. We noticed that CRISPR-mediated deletion or pharmacological inhibition of the MPC complex enhanced BTZ/CFZ-induced MM cell death with minimal impact on cell cycle progression. In fact, targeting the MPC complex compromised the bioenergetic capacity of MM cells, which is accompanied by reduced proteasomal activity, thereby exacerbating BTZ-induced cytotoxicity in vitro. Importantly, we observed that the RNA expression levels of several regulators of pyruvate metabolism were altered in advanced stages of MM for which they correlated with poor patient prognosis. Collectively, this study highlights the importance of the MPC complex for the survival of MM cells and their responses to proteasome inhibitors. These findings establish mitochondrial pyruvate metabolism as a potential target for the treatment of MM and an unappreciated strategy to increase the efficacy of proteasome inhibitors in the clinic.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•In murine AML models, Herc1 modulates resistance to nucleoside analogues and levels of Dck, the rate-limiting enzyme for their metabolism.•HERC1 is highly expressed in human AML and its elevated ...expression is associated with worse prognosis.
For several decades, induction therapy with nucleoside analogs, in particular cytarabine (Ara-C) and, to a lesser extent, fludarabine, has been the standard of care for patients diagnosed with acute myeloid leukemia (AML). Still, the anti-tumor efficacy of nucleoside analogs is often limited by intrinsic and acquired drug resistance, thereby leading to poor therapeutic response and suboptimal clinical outcomes. Here, we used genome-wide CRISPR-based pharmacogenomic screening to map the genetic factors that modulate the response to nucleoside analogs in AML and identified the E3 ubiquitin ligase Herc1 as a key modulator of Ara-C response in the MLL/AF9 (MA) and the HOXA9/MEIS1 (HM) murine AML models both in vitro and in vivo. Loss of HERC1 enhanced nucleoside analog-induced cell death in both murine and human AML cell lines by compromising cell cycle progression. In-depth proteomic analysis and subsequent validation identified deoxycytidine kinase Dck as a novel target of Herc1 in MA and HM murine cells. We observed that HERC1 is overexpressed in AML compared to other cancer types and higher HERC1 expression is associated with shorter overall survival of patients with AML in the TCGA and BEAT-AML cohorts. Collectively, this study highlights the importance of HERC1 in the response of AML cells to nucleoside analogs, thereby establishing this E3 ubiquitin ligase as a novel predictive biomarker and potential therapeutic target for the treatment of AML.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule regulating various cellular processes, including signal transduction, transcription, and cell survival. Although involved in ...inflammation and immune responses, its role in the germinal center reaction and B cell maturation remains unknown. We found that FHL2
mouse spleens displayed enlarged follicles with more B cells. When a T cell-dependent immune response was elicited using SRBC, FHL2
germinal center area was enhanced 2-fold compared with wild type (WT), concomitant with expanded dark zones. Nevertheless, the SRBC-induced rise in spleen IgG1 expression, and plasma IgG1 levels observed in WT were absent in FHL2
mice, and circulating plasma cells were also reduced in FHL2
This could be explained by deficient upregulation of spleen activation-induced cytidine deaminase mRNA. Interestingly, FHL2
B cells successfully underwent class-switch recombination in vitro, and both activation-induced cytidine deaminase induction and IgG1 response to SRBC were equivalent in B cell-deficient μMT mice transplanted with WT or FHL2
bone marrow, suggesting that the defects observed in FHL2
mice were not B cell intrinsic. However, spleen lysates from FHL2
mice revealed a disturbed spleen microenvironment, with reduced CXCL12 and CXCL13 levels compared with WT. Our data suggest that spleen FHL2 expression is essential for a normal germinal center reaction and proper induction of class-switch recombination in response to a T cell-dependent Ag, leading to the emergence of Ab producing plasma cells. This could be due to the regulation of spleen cytokine production by FHL2.
Genome-wide CRISPR screens have been extremely useful in identifying therapeutic targets in diverse cancers by defining genes that are essential for malignant growth. However, most CRISPR screens ...were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in 2 in vivo murine models of acute myeloid leukemia (AML) driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the major histocompatibility complex class I complex, Cd47, complement receptor Cr1l, and the β-4-galactosylation pathway. Importantly, several of these in vivo–specific hits have a prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as having in vivo–specific dependency with high prognostic relevance. Overall, we show an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo.
•In vivo CRISPR screens in AML define key interactors of the microenvironment, including integrins, immune modulators, and glycosylation.•Eight in vivo–specific hits are recurrently associated with adverse prognosis: BTBD6, FERMT3, ILK, SLC19A1, TAP2, TLN1, TPST2, and TRMT12.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen‐activated B cells, underpinning antibody affinity maturation and isotype ...switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert noncanonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B‐cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID‐dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof‐of‐concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract
Background and goal: RAD51D is a key player in DNA repair by homologous recombination (HR) and carriers of truncating RAD51D mutations have an increased risk for ovarian cancer (OC). ...However, the contribution of non-truncating RAD51D variants to cancer predisposition remains uncertain. We sought to fully characterize the previously described missense RAD51D variant c.620C>T;p.S207L in order to elucidate its role in OC.
Methods: A clinical panel screening was used to identify the RAD51D variant c.620C>T;p.S207L in two French Canadian (FC) kindred affected with familial High Grade Serous Cancer (HGSC) of the ovary or endometrium. High resolution melting, TaqMan genotyping and Sanger sequencing were used to genotype the p.S207L variant in a series of unselected cases of HGSC of the ovary and endometrium, breast, pancreas and colorectal cancer and healthy controls, all of a FC origin. Whole exome sequencing (WES) was performed to study the genetic signature characterizing RAD51D associated tumors. RAD51 foci formation and CRISPR-Cas9-stimulated and HR-mediated gene targeting assays were used to assess HR activity of RAD51D-S207L mutated CHO cells. HR activity in RAD51D-S207L mutated human cells was tested by a DR-GFP assay. The effect of RAD51D p.S207L on RAD51D-XRCC2 interactions was analyzed by co-immunoprecipitation and quantified in-vivo in a single cell colocalization assay. Sensitivity to PARP inhibitors (PARPi) was evaluated in a cell survival assay.
Results: Using deep sequencing and case-control genotyping studies, we showed that the missense RAD51D variant c.620C>T;p.S207L is over-represented in the French Canadian population affected by HGSC of the ovary (3.8% cases vs 0.002% controls; p < 0.0001).The frequency of the p.S207L variant did not differ from that of controls in breast, endometrial, pancreas and colorectal adenocarcinomas. A common haplotype shared by all the carriers suggested a founder origin for c.620C>T;p.S207L mutation. WES analysis of RAD51D tumor profiles revealed the presence of signature 3 which is known to be associated with HR defects. Functionally, we show that this mutation impairs HR by disrupting the RAD51D-XRCC2 interaction and confers PARP-inhibitor sensitivity to CHO cells.
Conclusions: This work identifies RAD51D p.S207L as the first bona fide pathogenic missense susceptibility allele for HGSC of the ovary and supports the use of targeted PARPi therapies in OC patients carrying missense RAD51D mutations.
Citation Format: Barbara Rivera, Massimo R. Di Iorio, Jessica Frankum, Javad Nadaf, Somayyeh Fahiminiya, Suzanna L. Arcand, David Burk, Damien Grapton, Eva Tomiak, Valerie Hastings, Nancy Hamel, Rabea Wagener, Olga Aleynikova, Sylvie Giroux, Fadi F. Hamdan, Alexandre Orthwein, George Zogopoulos, Francois Rousseau, Albert Berghuis, Diane M. Provencher, Guy A. Rouleau, Jacques L. Michaud, Anne-Marie Mes-Masson, Jacek Majewski, Susanne Bens, Reiner Siebert, Steven Narod, Mohammad Akbari, Chris J. Lord, Patricia N. Tonin, Alexandre Dionne-Laporte, William D. Foulkes. A functionally null RAD51D missense mutation is strongly associated with ovarian carcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2479. doi:10.1158/1538-7445.AM2017-2479
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