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Purpose:In clinical trials, the expansion and persistence of chimeric antigen receptor (CAR) T cells correlate with therapeutic efficacy. However, properties of CAR T cells that enable their in vivo proliferation have still to be consistently defined and the role of CAR T bag content has never been investigated in a real-life setting.Experimental Design:Residual cells obtained after washing 61 anti-CD19 CAR T product bags were analyzed to identify tisagenlecleucel/Tisa-cel and axicabtagene ciloleucel/Axi-cel phenotypic features associated with postinfusion CAR T-cell in vivo expansion and with response and survival.Results:While Tisa-cel was characterized by a significant enrichment in CAR+CD4+ T cells with central memory (P < 0.005) and effector (P < 0.005) phenotypes and lower rates of CAR+CD8+ with effector memory (P < 0.005) and naïve-like (P < 0.05) phenotypes as compared with Axi-cel, the two products displayed similar expansion kinetics. In vivo CAR T-cell expansion was influenced by the presence of CAR T with a CD8+ T central memory signature (P < 0.005) in both Tisa-cel and Axi-cel infusion products and was positively associated with response and progression-free survival (P < 0.05).Conclusions:Our data indicate that despite the great heterogeneity of Tisa-cel and Axi-cel products, the differentiation status of the infused cells mediates CAR T-cell in vivo proliferation that is necessary for antitumor response.

Supplementary Data from Phenotypic Composition of Commercial Anti-CD19 CAR T Cells Affects In Vivo Expansion and Disease Response in Patients with Large B-cell Lymphoma

Mature T-cell lymphomas (MTCLs) represent a heterogeneous group of aggressive non-Hodgkin lymphomas comprising different entities. Anthracycline-based regimens are considered the standard of care in the front-line treatment. However, responses to these approaches have been neither adequate nor durable, and new treatment strategies are urgently needed to improve survival. Genomic instability is a common feature of cancer cells and can be caused by aberrations in the DNA damage response (DDR) and DNA repair mechanisms. Consistently, molecules involved in DDR are being targeted to successfully sensitize cancer cells to chemotherapy. Recent studies showed that some hematological malignancies display constitutive DNA damage and intrinsic DDR activation, but these features have not been investigated yet in MTCLs. In this study, we employed a panel of malignant T cell lines, and we report for the first time the characterization of intrinsic DNA damage and basal DDR activation in preclinical models in T-cell lymphoma. Moreover, we report the efficacy of targeting the apical kinase ATM using the inhibitor AZD0156, in combination with standard chemotherapy to promote apoptotic cell death. These findings suggest that DDR is an attractive pathway to be pharmacologically targeted when developing novel therapies and improving MTCL patients’ outcomes.

Purpose: In clinical trials, the expansion and persistence of chimeric antigen receptor (CAR) T cells correlate with therapeutic efficacy. However, properties of CAR T cells that enable their in vivo proliferation have still to be consistently defined and the role of CAR T bag content has never been investigated in a real-life setting. Experimental Design: Residual cells obtained after washing 61 anti-CD19 CAR T product bags were analyzed to identify tisagenlecleucel/Tisa-cel and axicabtagene ciloleucel/Axi-cel phenotypic features associated with postinfusion CAR T-cell in vivo expansion and with response and survival. Results: While Tisa-cel was characterized by a significant enrichment in CAR+CD4+ T cells with central memory (P < 0.005) and effector (P < 0.005) phenotypes and lower rates of CAR+CD8+ with effector memory (P < 0.005) and naïve-like (P < 0.05) phenotypes as compared with Axi-cel, the two products displayed similar expansion kinetics. In vivo CAR T-cell expansion was influenced by the presence of CAR T with a CD8+ T central memory signature (P < 0.005) in both Tisa-cel and Axi-cel infusion products and was positively associated with response and progression-free survival (P < 0.05). Conclusions: Our data indicate that despite the great heterogeneity of Tisa-cel and Axi-cel products, the differentiation status of the infused cells mediates CAR T-cell in vivo proliferation that is necessary for antitumor response.

Summary Patients affected by lymphoid malignancies (LM) are frequently immune‐compromised, suffering increased mortality from COVID‐19. This prospective study evaluated serological and T‐cell responses after complete mRNA vaccination in 263 patients affected by chronic lymphocytic leukaemia, B‐ and T‐cell lymphomas and multiple myeloma. Results were compared with those of 167 healthy subjects matched for age and sex. Overall, patient seroconversion rate was 64·6%: serological response was lower in those receiving anti‐cancer treatments in the 12 months before vaccination: 55% vs 81·9% (P

Background Anthracycline-containing regimens, namely cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) or CHOEP (CHOP + etoposide), represent the current standard of care for patients with newly diagnosed peripheral T-cell lymphomas (PTCLs), although responses are unsatisfactory. In this study, we investigated the pathways able to mitigate the sensitivity to CHOP-based regimens in preclinical models of T-cell lymphoma (TCL) to select agents for the development of CHOP-based drug combinations. Methods We performed gene expression profiling of malignant T-cell lines exposed to CHOEP; flow cytometry was employed to study the effects of drug combinations on cell viability, cell cycle distribution, apoptosis and mitochondrial membrane depolarisation. Western blot analyses were performed to study cell signalling downstream of the T-cell receptor and apoptosis. The in vivo effect of the drug combination was tested in xenograft models. Results We highlighted a modulation of tyrosine kinases belonging to the T-cell receptor pathway upon chemotherapy that provided the rationale for combining the tyrosine kinase inhibitor dasatinib with CHOEP. Dasatinib improves CHOEP activity and reduces viability in vitro. Furthermore, combination treatment results in tumour growth inhibition in in vivo xenograft mouse models. Conclusions Our data provide the rationale for clinical testing of the dasatinib–CHOEP combination in patients with T-cell lymphoma.

Protein acetylation and deacetylation events are finely regulated by lysine-acetyl-transferases and lysine-deacetylases and constitute an important tool for the activation or inhibition of specific cellular pathways. One of the most important lysine-acetyl-transferases is p300, which is involved in the regulation of gene expression, cell growth, DNA repair, differentiation, apoptosis, and tumorigenesis. A well-known target of p300 is constituted by the tumor suppressor protein p53, which plays a critical role in the maintenance of genomic stability and whose activity is known to be controlled by post-translational modifications, among which acetylation. p300 activity toward p53 is negatively regulated by the NAD-dependent deacetylase SIRT1, which deacetylates p53 preventing its transcriptional activation and the induction of p53-dependent apoptosis. However, the mechanisms responsible for p53 regulation by p300 and SIRT1 are still poorly understood. Here we identify the nucleosome assembly protein TSPY-Like 2 (TSPYL2, also known as TSPX, DENTT, and CDA1) as a novel regulator of SIRT1 and p300 function. We demonstrate that, upon DNA damage, TSPYL2 inhibits SIRT1, disrupting its association with target proteins, and promotes p300 acetylation and activation, finally stimulating p53 acetylation and p53-dependent cell death. Indeed, in response to DNA damage, cells silenced for TSPYL2 were found to be defective in p53 activation and apoptosis induction and these events were shown to be dependent on SIRT1 and p300 function. Collectively, our results shed new light on the regulation of p53 acetylation and activation and reveal a novel TSPYL2 function with important implications in cancerogenesis.

Cell cycle and apoptosis regulator 2 (CCAR2 or DBC1) is a human protein recently emerged as a novel and important player of the DNA damage response (DDR). Indeed, upon genotoxic stress, CCAR2, phosphorylated by the apical DDR kinases ATM and ATR, increases its binding to the NAD+-dependent histone deacetylase SIRT1 and inhibits SIRT1 activity. This event promotes the acetylation and activation of p53, a SIRT1 target, and the subsequent induction of p53 dependent apoptosis. In addition, CCAR2 influences DNA repair pathway choice and promotes the chromatin relaxation necessary for the repair of heterochromatic DNA lesions. However, besides DDR, CCAR2 is involved in several other cellular functions. Indeed, through the interaction with transcription factors, nuclear receptors, epigenetic modifiers and RNA polymerase II, CCAR2 regulates transcription and transcript elongation. Moreover, promoting Rev-erbα protein stability and repressing BMAL1 and CLOCK expression, it was reported to modulate the circadian rhythm. Through SIRT1 inhibition, CCAR2 is also involved in metabolism control and, suppressing RelB and p65 activities in the NFkB pathway, it restricts B cell proliferation and immunoglobulin production. Notably, CCAR2 expression is deregulated in several tumors and, compared to the non-neoplastic counterpart, it may be up- or down-regulated. Since its up-regulation in cancer patients is usually associated with poor prognosis and its depletion reduces cancer cell growth in vitro, CCAR2 was suggested to act as a tumor promoter. However, there is also evidence that CCAR2 functions as a tumor suppressor and therefore its role in cancer formation and progression is still unclear. In this review we discuss CCAR2 functions in the DDR and its multiple biological activities in unstressed cells.

Background: CD19-directed CAR-T cell therapy has shown promising efficacy in relapsed/refractory (R/R) B-cell malignancies in clinical trials resulting in the approval and commercialization of two products (tisagenlecleucel/Tisa-cel and axicabtagene ciloleucel/Axi-cel) for R/R diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). However, relapses occur in 60-65% of patients (pts) and thus a better understanding of the early determinants of response is critical to improve long-term survival in the real-world scenario. Aims of the study: To assess whether CAR-T cell expansion after infusion represents a crucial determinant to sustain effective anti-tumor responses to both Tisa-cel and Axi-celTo evaluate differences in CAR-T cell kinetics due to the use of CD28 or 4-1BB costimulatory moleculesTo identify immune phenotypic features of infusion products accounting for CAR-T cell expansion and survival probability Methods: We analyzed samples from 43 pts [29 DLBCL, 8 high grade B-cell lymphoma (HGBCL) and 6 PMBCL] treated with Axi-cel (n=22) and Tisa-cel (n=21) at the Fondazione IRCCS Istituto Nazionale Tumori prospectively collected between November 2019 and April 2021. CAR-T cells were monitored in the peripheral blood (PB) on days 0, 4, 7, 10, 14, 21, 28 and monthly post infusion by flow cytometry (FCM). Cells were stained with CD19 CAR Detection Reagent (Miltenyi), CD3, CD4, CD8, CD45, CD14, CD45RO, CD62L, CD197, CD279, CD223 and CD366. Residual cells obtained from washings of 32 infused commercial CAR-T bags (10 Tisa-cel and 22 Axi-cel) were also analyzed by FCM. Data were acquired on a BD FACSCanto II (BD Biosciences) and a MACSQuant® Analyzer MQ10 (Miltenyi) and analyzed using FlowJo software, version 10. Results: The median time to maximal expansion of CAR-T cells was at day 10 post infusion with no differences between Axi-cel and Tisa-cel [median concentration at day 10 (C 10) 25 for Axi-cel vs 26 CAR-T cells/µl for Tisa-cel; p, ns], nor among the different histologies (median C 10 33 for DLBCL vs 19 for HGBCL vs 18 CAR-T cells/µl for PMBCL; p, ns). On the contrary, CAR-T peak concentration (C max) was higher in responders at 3 months post infusion (RE, n=28) (defined as pts achieving complete or partial response by PET/CT) than in non responders (NR, n=13) (median C max 87 in RE vs 26 in NR CAR-T cells/µl; p Conclusion: To the best of our knowledge, this is the first study assessing the clinical utility of early CAR-T cell monitoring in lymphoma pts receiving both commercial anti-CD19 CAR-T cell therapies. We provide evidence that in pts treated with Axi-cel and Tisa-cel: i) the in vivo kinetics of the CAR-T cell products are similar, consistent with the fact that no differences in the outcome of Axi-cel and Tisa-cel treated pts were detected; ii) CAR-T cell expansion is critical for efficacy and predicts the PFS; iii) circulating CAR-T cells in responders have a naïve phenotype; iv) a memory signature in the CAR-T cell product before infusion is associated with in vivo expansion and survival. Figure 1 Figure 1. Disclosures Chiappella: Celgene Bristol Myers Squibb: Other: lecture fee, advisory board; Incyte: Other: lecture fee; Novartis: Other: lecture fee; Astrazeneca: Other: lecture fee; Servier: Other: lecture fee; Takeda: Other: advisory board; Gilead Sciences: Other: lecture fee, advisory board; Clinigen: Other: lecture fee, advisory board; Roche: Other: lecture fee, advisory board; Janssen: Other: lecture fee, advisory board. Corradini: AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Consultancy; AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Honoraria; KiowaKirin; Incyte; Daiichi Sankyo; Janssen; F. Hoffman-La Roche; Kite; Servier: Consultancy; Novartis; Gilead; Celgene: Consultancy, Other: Travel and accommodations; BMS: Other: Travel and accommodation; Sanofi: Consultancy, Honoraria; Amgen; Takeda; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations; Incyte: Consultancy; Novartis, Janssen, Celgene, BMS, Takeda, Gilead/Kite, Amgen, AbbVie: Other: travel and accomodations.

Background: Chimeric antigen receptor (CAR) T-cell therapy is an effective treatment in approximately 40% of relapsed/refractory lymphomas. High rates of complete remission (CR) observed in clinical trials, led to fast-track FDA and EMA approval and commercialization of two CD19-directed CAR T-cell products (tisagenlecleucel/Tisa-cel/Kymriah and axicabtagene ciloleucel/Axi-cel/Yescarta) for refractory/relapsed diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). In these studies, CAR T-cell engraftment and in vivo expansion have a crucial impact on disease response and toxicity. It is of paramount importance to develop homogeneous standardized approaches for monitoring expansion and persistence of CAR T-cells also in the case of cells not detectable by flow cytometry (FCM) as these data can guide clinical decision making. Aims of this study were: to assess relevant time-points for FCM analysis across the different products to develop a unique quantitative PCR assay to detect both tisa-cel and axi-cel. Methods: We prospectively collected samples from 28 patients (pts) (16 DLBCL, 5 transformed follicular lymphoma and 7 PMBCL) treated with axi-cel (n=15) and tisa-cel (n=13) at the Fondazione IRCCS Istituto Nazionale Tumori between Nov 2019 and July 2020. CAR T-cells were monitored in the peripheral blood (PB) on days 0, 4, 7, 9, 14, 21, 28 and monthly post infusion by FCM. Cells were stained with CD19 CAR Detection Reagent (Miltenyi), Anti-Biotin-PE, CD3-FITC, CD4-VioGreen, CD8-APC-Vio770, CD45-VioBlue, CD14-APC and 7-AAD Staining Solution and analyzed by FCM using the MACSQuant® Analyzer (Miltenyi). To evaluate CAR T-cell persistence, we also generated a unique droplet digital PCR (ddPCR) primer-probe assay to target both tisagen-cel and axi-cel. We obtained a partial cDNA sequence of the two CAR constructs by amplification and direct sequencing of fragments generated with primers designed by educated guess to bind to the anti-CD19 murine single-chain variable fragment (scFV) and to the CD8α or CD28 trans-membrane regions of tisagen-cel and axi-cel vectors respectively. The assay was used to quantify CAR+ cells on genomic DNA extracted from PB cells collected serially after infusion. FCM and molecular data were correlated to outcome. Results: Peak expansion of CAR T-cells by FCM occurred within the first 14 days post-infusion. At the point of maximal expansion, collectively a mean of 17% of circulating CD3+ T cells were CAR+ and CAR+ cells were more abundant among CD3+ cells in pts receiving axi-cel (26% vs 8.5% for tisagen-cel, p62/ul was prognostic with sensitivity of 67% and specificity of 76% (Receiver operating characteristic analysis). At subsequent time points the number of CAR T-cells decreased, but continued to be detectable by FCM up to 6 months post infusion although at very low levels (mean 2 CAR+ cell/ul) irrespective of outcome. To evaluate whether these low counts reliably represent CAR T+ cells or are rather signals due to aspecific binding of the antibodies, a ddPCR assay was set up. The assay displayed an excellent separation between positive and negative droplets on both axi-cel and tisagen-cel products, good reproducibility and specificity with max standard deviation of 0.06 and no CAR positive signal in healthy donors. Additionally, the CAR specific signal was detectable up to the 0,01% dilution. FCM and ddPCR data had a good correlation (r=0.93, p Conclusion: The combined use of FCM and the novel molecular assay facilitates the precise enumeration of commercial anti-CD19 CAR T-cells. Higher frequencies of CAR+ T cells at day 9 distinguish responders irrespectively of the product they received in our small prospective series. Updated results will be presented. Disclosures Chiappella: Servier: Honoraria; Roche: Honoraria; Takeda: Honoraria; Iqone: Honoraria; Janssen: Honoraria; Gilead-Kite: Honoraria; Celgene: Honoraria. Corradini:Kite: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Incyte: Consultancy; BMS: Other; Takeda: Consultancy, Honoraria, Other; Servier: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; F. Hoffman-La Roche Ltd: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Travel and accommodations paid by for; KiowaKirin: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel and accommodations paid by for; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Gilead: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Daiichi Sankyo: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Other: Travel and accommodations paid by for.

Historically, the differential diagnosis between different nodal peripheral T-cell lymphoma (PTCL) subtypes based on morphological and phenotypic grounds has posed great challenges. In the last few years, our knowledge of the molecular bases of different PTCLs has significantly expanded. However, peripheral T-cell lymphomas not otherwise specified (PTCL-NOSs) are still regarded to as a heterogeneous category encompassing PTCL cases not fitting other, more homogeneous, subtypes. In fact, PTCL-NOS is one of the few lymphoma subtypes where no recurrent driver mutations have been reported so far. In order to better characterized the PTCL-NOS genomic landscape, we decided to investigate 11 PTCL-NOS patients by a whole genome sequencing (WGS) approach (median coverage 27X). Ten out of eleven samples were collected from FFPE blocks and 2 were removed from analysis: one due to low cancer cell fraction (CCF) and the other based on cluster generation issues during sequencing likely caused by a hyper-fragmented DNA. Among the remaining 9 cases, we extracted 59,617 somatic base substitutions (range 2,471-10,756, median 6,358 per patient) and 20,531 small insertion-deletions (indels) (range 84-6,397, median 1,580). We were able to characterize the spectrum of FFPE-induced artefacts, mostly composed of point mutations and indels within LINE-1 (L1) elements, predominantly of the L1PA family. This is a crucial quality control step that could be applied to similar future studies from archive samples. Four samples were heavily involved by FFPE-related artefacts and were excluded for this reason. Using a non-negative matrix factorization (NNMF) algorithm we investigated for the first time the PTCL-NOS mutational signature landscape. We did not find novel processes in this entity, but rather known processes operative in other lymphoid malignancies. Among those: signatures 1 and 5, deriving from the age-related process of spontaneous deamination of methylated cytosines; signatures 2 and 13 deriving from aberrant activity of the APOBEC family of DNA deaminases; signatures 17 and 8, pertaining to two yet poorly characterized processes. The contribution of different processes to the mutational spectrum of each case was profoundly heterogeneous. Combining our data set with 64 previously published whole exome sequencing cases (23 ALCL, 15 AITLs, 9 PTCL-NOSs and 16 EATL-II), we confirmed the lack of recurrent driver mutations among PTCL-NOS. Taking advantage of WGS data, we therefore focused on structural variants (SVs: inversions, translocations, internal tandem duplications and deletions) and copy number alterations (CNAs). We found 372 SVs, with a stunning median of 73 per sample (range 56-86). Even more interesting, at least one complex event was observed in all but one patients, including one whole genome duplication (WGD) and five chromothripsis events in three patients, suggesting a critical role of SVs in shaping the PTCL-NOS genome. We found that known onco-drivers were recurrently disrupted by such events: the most frequent target was CDKN2A, deleted in 4 out of 5 patients, 2 of which carried homozygous deletions. Interestingly, PTEN loss was observed in 2 out of 4 CDKN2A-deleted patients. Given the high prevalence of these deletions, we extended our observation to an independent validation set of ALCLs (n=56), AITL (n=22) and PTCL-NOS (n=59) investigated by FISH (n=36), next generation sequencing (n=25) or SNP6 array series (n=76). Overall, CDKN2A was deleted in 22/59 (37%) PTCL-NOSs cases, and in 17/22 (77%) both alleles were lost. PTEN was deleted in 12/59 (20%) PTCL-NOS cases, all of which also carried a CDKN2A loss. Strikingly, the co-occurrence of CDKN2A and PTEN was found only among PTCL-NOS, and in none of the other entities. With the limitations of the small sample size, the presence of CDKN2A bi-allelic deletions was associated with inferior survival (25% [95% CI: 9-66%] 5-y OS for deleted cases vs 52% [95% CI: 28-96%] for wt/hemizygous cases, p=0.042) among patients treated with an autologous bone marrow transplant front line program for advance stage and high-risk disease (n=19). Our observations point at SVs as a main driver of PTCL-NOS, often involving known cancer genes and their downstream pathways. Furthermore, our data highlighted recurrent gene deletions that may be relevant for differential diagnosis within this category of lymphomas. Disclosures Bolli: Celgene: Honoraria. Chiappella:Roche: Other: lecture fees; Amgen: Other: lecture fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Nanostring: Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Teva: Other: lecture fees. Corradini:Celgene: Honoraria, Other: Advisory Board & Lecturer; Novartis: Honoraria, Other: Advisory Board & Lecturer; Roche: Honoraria, Other: Advisory Board & Lecturer; Sanofi: Honoraria, Other: Advisory Board & Lecturer; Gilead: Honoraria, Other: Advisory Board & Lecturer; Sandoz: Other: Advisory Board; Abbvie: Honoraria, Other: Advisory Board & Lecturer; Takeda: Honoraria, Other: Advisory Board & Lecturer; Amgen: Honoraria, Other: Advisory Board & Lecturer; Janssen: Honoraria, Other: Lecturer.

Peripheral T-cell lymphomas (PTCLs) represent a rare and heterogeneous group of aggressive non-Hodgkin's lymphomas comprising different entities. Anthracycline-based regimens (usually CHOP, cyclophosphamide/doxorubicin/vincristine/prednisone) followed by stem cell transplantation are considered the standard of care in the front-line setting. However, long-term disease control can be achieved only in 30-40% of young patients. Increasing evidences highlight the role of tumor microenvironment in sustaining tumor progression and aggressiveness. Among microenvironment components, a central role is played by cancer associated fibroblast (CAFs). Recent studies demonstrated that this is applicable not only to solid tumors but also to haematological malignancies. Here, we report for the first time the isolation and characterization of PTCL-CAFs and their role in promoting cell proliferation. We kept in culture a skin biopsy of a cutaneous localization of a peripheral T cell lymphoma. Fibroblast-like cells grown out from the biopsy were isolated, cultivated for few passages and then characterized. Flow cytometric analyses revealed that the cell population entirely expressed cell-surface antigens specific for fibroblasts, such as CD140b and CD90, while they did not express CD45 and CD31, thus excluding hematopoietic or endothelial origin. Biochemical analyses confirmed that all cells were positive for vimentin, while αSMA expression highlighted the activated status of the cells isolated. We then assessed the in vitro effect of CAFs on PTCL cell growth by co-colture experiments. For this purpose, we coltured for 8 days the PTCL cell line OCI-Ly12 in fresh medium or in medium previously conditioned by CAFs or normal skin fibroblasts (NFs). PTCL cell growth was not affected by co-colture either with CAFs or with NFs. Moreover, the presence of conditioned medium did not alter the response of OCI-Ly12 cells to CHOP treatments. We then assessed the in vivo effect of PTCL-CAFs. We subcutaneously injected CAFs alone, OCI-Ly12 cells alone and OCI-Ly12 together with CAFs (ratio 1:1) in NOD/SCID mice and monitored tumor growth for 20 days. In contrast to the effect observed in vitro, co-injection of CAFs with cancer cells substantially promoted tumor growth (tumor volume OCI-Ly12 0,87±0,47 cm3; OCI-Ly12+CAFs 2,30±0,26 cm3). As expected, CAFs alone did not develop a measurable mass. Subsequent immunohistochemical staining with CD31 revealed the presence of more vascular structures in tumors originated by OCI-Ly12+CAFs co-injection than by OCI-Ly12 alone, suggesting that CAFs presence sustains tumor growth by promoting angiogenesis. In summary, we show that PTCL-derived CAFs promote cancer cell growth in vivo, while leaving unaffected cell growth in vitro and the response to chemotherapy. This is the first evidence of the role played by the microenvironment in promoting malignant cell proliferation in PTCLs. Citation Format: Martina Magni, Sara Rizzitano, Alessio Pellegrinelli, Paolo Corradini, Cristiana Carniti. Peripheral T cell lymphoma-associated fibroblasts promote tumor growth in an in vivo model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3123.

// Martina Magni 1 , Vincenzo Ruscica 1,3 , Michela Restelli 1 , Enrico Fontanella 1 , Giacomo Buscemi 2 and Laura Zannini 1 1 Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2 Department of Biosciences, University of Milan, Milan, Italy 3 Current address: Max Planck Institute for Developmental Biology, Tubingen, Germany Correspondence to: Laura Zannini, email: // Keywords : DNA damage, DNA repair, phosphorylation, chromatin relaxation Received : May 29, 2015 Accepted : May 29, 2015 Published : June 10, 2015 Abstract Cell cycle and apoptosis regulator 2 (CCAR2, formerly known as DBC1) is a nuclear protein largely involved in DNA damage response, apoptosis, metabolism, chromatin structure and transcription regulation. Upon DNA lesions, CCAR2 is phosphorylated by the apical kinases ATM/ATR and this phosphorylation enhances CCAR2 binding to SIRT1, leading to SIRT1 inhibition, p53 acetylation and p53-dependent apoptosis. Recently, we found that also the checkpoint kinase Chk2 and the proteasome activator REGγ are required for efficient CCAR2-mediated inhibition of SIRT1 and induction of p53-dependent apoptosis. Here, we report that CCAR2 is required for the repair of heterochromatic DNA lesions, as cells knock-out for CCAR2 retain, at late time-points after genotoxic treatment, abnormal levels of DNA damage-associated nuclear foci, whose timely resolution is reinstated by HP1β depletion. Conversely, repair of DNA damages in euchromatin are not affected by CCAR2 absence. We also report that the impairment in heterochromatic DNA repair is caused by defective Chk2 activation, detectable in CCAR2 ablated cells, which finally impacts on the phosphorylation of the Chk2 substrate KAP1 that is required for the induction of heterochromatin relaxation and DNA repair. These studies further extend and confirm the role of CCAR2 in the DNA damage response and DNA repair and illustrate a new mechanism of Chk2 activity regulation. Moreover, the involvement of CCAR2 in the repair of heterochromatic DNA breaks suggests a new role for this protein in the maintenance of chromosomal stability, which is necessary to prevent cancer formation.

Human DBC1 (Deleted in Breast Cancer 1; KIAA1967; CCAR2) is a protein implicated in the regulation of apoptosis, transcription and histone modifications. Upon DNA damage, DBC1 is phosphorylated by ATM/ATR on Thr454 and this modification increases its inhibitory interaction with SIRT1, leading to p53 acetylation and p53-dependent apoptosis. Here, we report that the inhibition of SIRT1 by DBC1 in the DNA damage response (DDR) also depends on Chk2, the transducer kinase that is activated by ATM upon DNA lesions and contributes to the spreading of DNA damage signal. Indeed we found that inactivation of Chk2 reduces DBC1-SIRT1 binding, thus preventing p53 acetylation and DBC1-induced apoptosis. These events are mediated by Chk2 phosphorylation of the 11S proteasome activator REGγ on Ser247, which increases REGγ-DBC1 interaction and SIRT1 inhibition. Overall our results clarify the mechanisms underlying the DBC1-dependent SIRT1 inhibition and link, for the first time, Chk2 and REGγ to the ATM-DBC1-SIRT1 axis.

Human CCAR2 has recently emerged as having a pivotal role in the DNA damage response, promoting apoptosis and repair of heterochromatic DNA breaks. However, less is known about the function of CCAR2 in tumor formation and cancer progression. Here, we demonstrate, for the first time, that CCAR2 loss inhibits the proliferation of cancer cells, but preserves the growth of normal cells. Investigating the mechanisms responsible for this differential effect, we found that CCAR2 depletion specifically impairs the activation of AKT pathway in cancer cells, but not in normal cells, by reducing AKT phosphorylation on Ser473. This effect is achieved through the transcriptional upregulation of TRB3 gene and accumulation of TRB3 protein, which then binds to and inhibits the phosphorylation and activation of AKT. The defective activation of AKT finally results in reduced GSK3β phosphorylation, prevention of G1/S transition and inhibition of cancer cell growth. These results establish an important role for CCAR2 in cancer cells proliferation and could shed new light on novel therapeutic strategies against cancer, devoid of detrimental side effects.