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Multiple myeloma (MM) is a malignancy of plasma cells (PC) that grow within the bone marrow and maintain massive immunoglobulin (Ig) production. Disease evolution is driven by genetic lesions, whose effects on cell biology and fitness underlie addictions and vulnerabilities of myeloma cells. Several genes mutated in myeloma are strictly involved in dictating PC identity and antibody factory function. Here, we evaluate the impact of mutations in IRF4, PRDM1, and XBP1, essential transcription factors driving the B to PC differentiation, on MM cell biology and homeostasis. These factors are highly specialized, with limited overlap in their downstream transcriptional programs. Indeed, IRF4 sustains metabolism, survival, and proliferation, while PRDM1 and XBP1 are mainly responsible for endoplasmic reticulum expansion and sustained Ig secretion. Interestingly, IRF4 undergoes activating mutations and translocations, while PRDM1 and XBP1 are hit by loss-of-function events, raising the hypothesis that containment of the secretory program, but not its complete extinction, may be beneficial to malignant PCs. Finally, recent studies unveiled that also the PRDM1 target, FAM46C/TENT5C, an onco-suppressor uniquely and frequently mutated or deleted in myeloma, is directly and potently involved in orchestrating ER homeostasis and secretory activity. Inactivating mutations found in this gene and its interactors strengthen the notion that reduced secretory capacity confers advantage to myeloma cells. We believe that dissection of the evolutionary pressure on genes driving PC-specific functions in myeloma will disclose the cellular strategies by which myeloma cells maintain an equilibrium between antibody production and survival, thus unveiling novel therapeutic targets.

Background Multiple Myeloma (MM) is a disease driven by numerous genetic and epigenetic alterations, however true drivers of the disease have yet to be identified. Methods To identify new dependencies and actionable therapeutic targets in MM we integrated gene expression and genetic dependency (CRISPR KO). Results We found that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among others, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well and established MM compared to normal plasma cells (NPC). Loss-of-function studies using shRNA, siRNAs as well as antisense GapMers specific for BOB-1 confirmed significant impact on MM cell viability. Transcriptomic analysis by RNA-sequencing revealed a small set of genes commonly modulated in MM cell lines upon BOB-1 depletion, including the XBP1- BHLHA15 axis involved in lipid synthesis and unfolded protein response (UPR). Interestingly, among the genes most significantly upregulated by BOB-1 depletion was heme oxygenase 1 (HMOX1), that was affected via the NRF2/Keap1 pathway. We observed that HMOX1 expression is significantly lower in MM cells from patients compared to normal plasma cells and correlates with poor clinical outcome, suggesting important role in MM. Moreover, we found that siRNA depletion of HMOX1 reverted the inhibition of MM cell growth caused by BOB-1 KD, confirming significant role for HMOX1 in the BOB-1 addiction observed in MM cells. Next, we performed gene set enrichment analysis (GSEA) and observed ribosome biogenesis pathways and mRNA translation and elongation processes, along with WNT and senescence pathways, to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. In MM cell lines, BOB-1 knockdown decreased de novo protein synthesis, while its overexpression significantly enhances protein synthesis compared to control cells. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular kappa and lambda light chains production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result, BOB-1 depletion was associated with induction of resistance to proteasome inhibition. Conclusion In conclusion, we report BOB1 as a specific dependency in MM cells with potential role on modulating the protein load/capacity balance in MM cells and therefore the sensitivity to proteasome inhibition.

Background Protein secretion is a driving force in evolution and tissue specialization. Plasma cells (PC), responsible for intense antibody (Ab) production, are paradigmatic professional secretors whose differentiation entails rapid endoplasmic reticulum (ER) expansion and concerted expression of the protein translocation, folding and trafficking machinery. A novel player in PC differentiation, recently implicated in Ab immunity is the non-canonical poly(A) polymerase FAM46C/TENT5C. Methods In developing PCs, FAM46C is induced to polyadenylate and enhance the translation of a number of mRNAs encoding Igs and ER-targeted proteins [1,2]. Being deleted or mutated in up to 20% multiple myeloma (MM) patients, but intact in other cancers, the FAM46C gene is among the most frequent genomic hits in MM and a myeloma-specific onco-suppressor [3,4]. Results We have recently shown that the ER-specific action of FAM46C depends on its interaction with the ER transmembrane FNDC3 proteins. As a result, FAM46C concertedly boosts the expression of ER and Golgi proteins, potently upsizing the secretory apparatus and Ig secretion [5]. Our data suggest that in MM the FAM46C gene is under selective pressure to contain the proteosynthetic, oxidative and metabolic stress associated with Ig secretion, favoring myeloma cell survival and growth. In keeping with this, exogenous re-expression of FAM46C in mutated MM cells raised Ig secretory capacity beyond sustainability, causing ATP shortage, ROS accumulation and apoptosis. Interestingly, although able to increase the secretory capacity across different cell types, FAM46C overexpression induced apoptosis exclusively in MM cells, being well tolerated in non-professional secretors, suggesting a key role of the secretory cargo in FAM46C toxicity. Moreover, we discovered a broader function of FAM46C in ER homeostasis beyond mRNA stabilization. Indeed, we found that FAM46C is part of a novel integrated network coordinating the biogenesis of ribonucleoprotein complexes, ER protein translation and import, and ER expansion, to harmonize massive Ab production with protein homeostasis. Finally, our proteomic analysis unveiled a surprising opposite modulation of signal recognition particle (SRP) proteins mediated by FAM46C. Indeed, while 4 out of 6 SRP proteins were positively regulated by FAM46C, the heterodimeric members SRP9 and SRP14, mediating translational arrest, increased upon FAM46C loss. Conclusions We infer that PCs have the ability to sense FAM46C-induced ER expansion and modulate SRP composition to minimize translational pausing and maximize Ab manufacture. This novel integrated network offers a framework to identify unprecedented highly specific therapeutic targets against PC dyscrasias.

FAM46C is a non-canonical poly(A) polymerase uniquely mutated in up to 20% of multiple myeloma (MM) patients, implying a tissue-specific tumor suppressor function. Here, we report that FAM46C selectively stabilizes mRNAs encoding endoplasmic reticulum (ER)-targeted proteins, thereby concertedly enhancing the expression of proteins that control ER protein import, folding, N-glycosylation, and trafficking and boosting protein secretion. This role requires the interaction with the ER membrane resident proteins FNDC3A and FNDC3B. In MM cells, FAM46C expression raises secretory capacity beyond sustainability, inducing ROS accumulation, ATP shortage, and cell death. FAM46C activity is regulated through rapid proteasomal degradation or the inhibitory interaction with the ZZ domain of the autophagic receptor p62 that hinders its association with FNDC3 proteins via sequestration in p62+ aggregates. Altogether, our data disclose a p62/FAM46C/FNDC3 circuit coordinating sustainable secretory activity and survival, providing an explanation for the MM-specific oncosuppressive role of FAM46C and uncovering potential therapeutic opportunities against cancer. Fucci et al. show that the poly(A) polymerase FAM46C acts as a multiple myeloma-specific tumor suppressor, increasing secretory capacity and antibody production beyond sustainability via its interaction with endoplasmic reticulum transmembrane FNDC3 proteins. Moreover, its activity is restricted through proteasomal degradation or p62-dependent aggregation and sequestration from FNDC3 proteins.

High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

Multiple Myeloma (MM) is a malignancy driven by numerous genetic and epigenetic alterations. Recurrent IgH translocations, somatic mutations and copy number abnormalities all contribute to myelomagenesis, however true drivers of the disease have not been well defined. To identify new targetable dependencies in MM, we generated high-quality active enhancer landscape using large cohort of primary patient myeloma cells (n=70), MM cell lines, and normal plasma cells. We integrated this data with an in-house curated atlas of 600+ active enhancer profile across a wide range of tumor types and normal tissues. Combining these data with gene expression and genetic dependency (CRISPR KO) enabled a multidimensional integration of how transcriptional regulation intersects with tumor specific dependencies. We identified that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among these, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well as symptomatic MM compared to normal plasma cells. To functionally validate the role of BOB-1 in MM, we performed loss-of-function studies using shRNA, siRNAs and antisense GapMers specific for BOB-1 and observed a significant impact on MM cell viability and cell cycle arrest. Transcriptomic analysis upon BOB-1 depletion by RNA-sequencing revealed a small set of genes commonly modulated in all 3 MM cell lines tested including the plasma cell differentiation related transcription factor XBP1 and heme oxygenase (HMOX1). Importantly, we observed ribosome biogenesis, RNA polymerase 1A transcription and mRNA translation and elongation processes to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Bob1 KD resulted in a rapid and robust decrease in the level of transcription of rDNA by RNA polymerase I as determined by qRT-PCR quantification of pre-rRNA (47S). In addition, ChiP assay revealed decreased binding of RNA polymerase 1A to the 18S ribosomal DNA promoter region in BOB-1 depleted cells compared to control. These data indicate that BOB1 downregulation results in the suppression of RNA-polymerase I activity in MM cells. RNA Pol I-dependent transcription governs abundance of rRNA and directly regulates cellular translational and proliferative capacity. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. We observed that in MM cells compared to control cells, BOB-1 KD decreased, while its overexpression significantly enhanced de novo protein synthesis. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular light chains (kappa or lambda) production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result of decreased protein production, BOB-1 depletion was associated with induction of resistance to proteasome inhibition suggesting that high expression of BOB-1 may be one the factors driving the exquisite sensitivity of MM cells to proteasome inhibitor. Interestingly, mass spectrometry analysis revealed BOB1 in a protein complex with mTOR, Raptor and mLST8 proteins which are members of mTORC1 complex which is also involved in ribosomal function and may suggest the mechanism of action of Bob-1 at molecular level. In conclusion, here we report BOB1 as a specific proximal dependency in MM cells with potential role in modulating the protein load/capacity balance and ribosomal biogenesis essential for MM cell protein production function and therefore their sensitivity to proteasome inhibition. Disclosures Shirasaki: FIMECS: Consultancy. Mitsiades: BMS: Research Funding; Nurix: Research Funding; H3 Biomedicine: Research Funding; Novartis: Research Funding; Abbvie: Research Funding; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Karyopharm: Research Funding; Sanofi: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals: Consultancy, Honoraria. Hajek: BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria. Munshi: Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Pfizer: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Novartis: Consultancy.

Multiple myeloma (MM) is a complex and heterogenous disease which is dependent on the surrounding microenvironment for growth and survival. In MM, dysregulation of transcriptional control is a major driver of tumor transformation and progression. To evaluate transcriptional programs activated in MM cells in the context of the bone marrow milieu, we have performed extensive transcriptomic analysis by RNA-seq and ATAC-seq using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines. We observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 cells significantly downregulated expression of Inhibitor of DNA Binding 2 (ID2), while footprint analysis of the open chromatin regions in MM cells upon interaction with BMSC revealed enrichment for binding motifs of the TCF family of transcription factors (E proteins). Inhibitors of DNA binding (ID) proteins control crucial transcriptional programs in B cell maturation via their heterodimerization with E proteins which are members of the basic helix-loop-helix (bHLH) class I family of transcription factors, repressing their DNA binding and therefore transcriptional activity. We found that ID2 expression is significantly lower in primary CD138+ MM cells from patients compared to normal plasma cells (NPCs). Moreover, we have previously implicated the B-cell factor TCF3 as a novel MM dependency. Using MM cell lines and primary samples, we observed elevated enhancer activity at TCF3 locus in primary malignant plasma cells compared to NPCs, which resulted in significant upregulation of TCF3 expression in MM patients. We also showed that TCF3 is regulated by a large proximal enhancer that is bound by MYC and is highly sensitive to chemical perturbation of enhancer co-activators such as BRD4. Genetic perturbation of TCF3 confirmed its critical role on MM cell growth and viability especially in IgH MYC translocated MM cell lines. We here further explored the role of ID2-TCF3 axis and the hypothesis that lower expression of ID2 drives higher TCF3 activity in MM cells, which is further enhanced in presence of the bone marrow microenvironment. Genetic modulation of ID2 significantly affected MM cell viability, with MM cells ectopically expressing ID2 displaying a cell growth arrest even in the presence of the supportive BM milieu. To define the mechanism of the observed oncosuppressive role of ID2 in MM, which is in line with preliminary observations in other hematological malignancy but in contrast with the pro-tumoral role described in solid tumors, we first performed immunoprecipitation of ID2 followed by mass spectrometry in 3 MM cell lines, and identified a very consistent and specific interaction with E proteins TCF3 and TCF12. Next, to explore the transcriptional programs dependent on ID2 we performed RNA-seq of 2 MM cell lines after ID2 overexpression. In line with our in vitro data, gene ontology and gene set enrichment analysis showed a significant downregulation of genes involved in E2F pathway, cell cycle progression and regulation of gene transcription. Interestingly, among the known TCF3 targets in B cells, only XBP-1 was significantly downregulated in MM cells after ID2 overexpression, suggesting the existence of a cell-specific TCF3 dependent transcriptional program in MM. Indeed, ATAC-seq experiments revealed ID2 overexpression led to a significant decrease of TCF3 binding motifs in open chromatin regions, confirming the relevance of ID2 in regulating TCF3 transcriptional activity in MM. In conclusion, while both E and ID proteins have been implicated in malignant transformation, their role in supporting MM transcriptional deregulation and tumor growth in the context of the microenvironment is being defined. Here, we have identified ID2 as a major regulator of the TCF3 dependent transcriptional program in MM, whose downregulation is essential to maintain MM proliferation and to mediate the benefits induced by MM-stroma interaction. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy.

Background Mitochondria are enticing potential targets against cancer, owing to their role as signaling hubs orchestrating key homeostatic functions. Of special interest is ClpP, a resident mitochondrial protease suggested to maintain OXPHOS by degrading damaged protein components and regulating the assembly of mito-ribosomes. While the exact role of ClpP in mammals remains unclear, its manipulation has been shown to induce leukemic cell death. Prompted by its distinctive expression in malignant plasma cells (PCs), we investigated the role of ClpP in maintaining mitochondrial and cellular homeostasis in multiple myeloma (MM) cells and tested it as a possible anti-myeloma target. Methods We analyzed the expression of ClpP mRNA in public and proprietary datasets of normal and malignant PCs and MM cell lines. We performed stable shRNA-mediated knockdown of ClpP (ClpPkd) in MM cell lines and analyzed its sequelae combining electron microscopy, Seahorse and ATP assays, transcriptomics, proteomics, and metabolomics. A proteolytically inactive ClpP mutant (ClpPmut) was expressed to entrap ClpP substrates for subsequent mass spectrometry identification and wet validation. Results ClpP mRNA was significantly higher in bone marrow-purified malignant vs. normal PCs, and MM cells were the highest ClpP-expressing human cancer cell lines. Attesting to a crucial role in myeloma, ClpPkd MM cell lines disappeared from culture due to rapid onset of cell cycle arrest and apoptosis. Intriguingly, toxicity in MM proved independent of the currently acknowledged ClpP-controlled mitochondrial functions, i.e., mito-ribosome assembly and OXPHOS maintenance. Indeed, Seahorse demonstrated different bioenergetics across MM lines, ranging from mixed oxidative/glycolytic to almost exclusively glycolytic. Yet, ClpPkd failed to abate ATP in glycolytic MM lines, but proved equally toxic across all lines, thus unveiling an energy-independent vulnerability. To unbiasedly define the role of ClpP in MM, we undertook a threefold orthogonal approach employing RNA-seq, proteomics, and metabolomics. Their integrated analysis upon ClpPkd revealed an unexpected impact of ClpPkd on protein translation in the cytosol via the processing of nuclear-encoded RNA, coupled with metabolic changes indicative of impaired fatty acid oxidation and pentose phosphate pathway. Finally, mass spectrometry of ClpPmut-entrapped partners identified myeloma-specific mitochondrial substrates, including chaperones and enzymes involved in RNA metabolism and oxidative stress. RNA-seq analyses are further characterizing the pathways impacted by ClpPkd and guiding wet validation experiments. Conclusions Overall, our data strongly suggest that ClpP is vital to MM cells due to a novel non-bioenergetic function, and that its manipulation is lethal via a broad perturbation of mitochondrial and cellular homeostasis.

Infections are a major cause of morbidity and mortality in hematological patients. We prospectively tested a new molecular assay (Verigene®) in 79 consecutive hematological patients, with sepsis by gram-negative bacteria. A total of 82 gram-negative microorganisms were isolated by blood cultures, of which 76 cases were mono-microbial. Considering the bacteria detectable by the system, the concordance with standard blood cultures was 100%. Resistance genes were detected in 20 of the isolates and 100% were concordant with the phenotypic antibiotic resistance. Overall, this new assay correctly identified 66/82 of all the gram-negative pathogens, yielding a general sensitivity of 80.5%, and providing information on genetic antibiotic resistance in a few hours. This new molecular assay could ameliorate patient management, resulting in a more rational use of antibiotics.

The major cause of death after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for acute myeloid leukemia (AML) is disease relapse. We investigated the expression of Inhibitory Receptors (IR; PD-1/CTLA-4/TIM-3/LAG-3/2B4/KLRG1/GITR) on T cells infiltrating the bone marrow (BM) of 32 AML patients relapsing (median 251 days) or maintaining complete remission (CR; median 1 year) after HSCT. A higher proportion of early-differentiated Memory Stem (TSCM) and Central Memory BM-T cells express multiple IR in relapsing patients than in CR patients. Exhausted BM-T cells at relapse display a restricted TCR repertoire, impaired effector functions and leukemia-reactive specificities. In 57 patients, early detection of severely exhausted (PD-1+Eomes+T-bet−) BM-TSCM predicts relapse. Accordingly, leukemia-specific T cells in patients prone to relapse display exhaustion markers, absent in patients maintaining long-term CR. These results highlight a wide, though reversible, immunological dysfunction in the BM of AML patients relapsing after HSCT and suggest new therapeutic opportunities for the disease., Allogeneic hematopoietic cell transplantation is the standard treatment of acute myeloid leukemia, but many patients relapse. Here the authors show increased markers of exhaustion and cancer antigen specificity within bone marrow-residing memory T cells precede and potentially predict the relapse.

Purpose: p21-activated kinase 4 (PAK4) plays a significant biological and functional role in a number of malignancies, including multiple myeloma (MM). On the basis of our promising findings in MM, we here characterize PAK4 expression and role in WM cells, as well effect of dual PAK4-NAMPT inhibitor (KPT-9274) against WM cell growth and viability. Experimental Design: We have analyzed mRNA and protein expression levels of PAK4 in WM cells, and used loss-of-function approach to investigate its contribution to WM cell viability. We have further tested the in vitro and in vivo effect of KPT-9274 against WM cell growth and viability. Results: We report here high-level expression and functional role of PAK4 in WM, as demonstrated by shRNA-mediated knockdown; and significant impact of KPT-9274 on WM cell growth and viability. The growth inhibitory effect of KPT-9274 was associated with decreased PAK4 expression and NAMPT activity, as well as induction of apoptosis. Interestingly, in WM cell lines treated with KPT-9274, we detected a significant impact on DNA damage and repair genes. Moreover, we observed that apart from inducing DNA damage, KPT-9274 specifically decreased RAD51 and the double-strand break repair by the homologous recombination pathway. As a result, when combined with a DNA alkylating agents bendamustine and melphalan, KPT-9274 provided a synergistic inhibition of cell viability in WM cell lines and primary patient WM cells in vitro and in vivo. Conclusions: These results support the clinical investigation of KPT-9274 in combination with DNA-damaging agent for treatment of WM.

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.

Background. Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML); nonetheless, relapse remains the major cause of death after such therapeutic option. Patients and Methods . We investigated the expression of Inhibitory Receptors (IR; i.e. PD-1, CTLA-4, TIM-3, LAG-3 and KLRG1) on different T-cell subsets infiltrating the bone marrow (BM) of 8 healthy donors (HD) and 32 allogeneic HSCT recipients diagnosed with Acute Myeloid Leukemia, collected at relapse (median 251 days) or at complete remission (CR) 1 year after HSCT. Inclusion criteria were: a diagnosis of acute myeloid leukemia or myelodysplastic syndrome, a relapse-free survival of at least 4 months after allogenein HSCT, absence of active GvHD, CMV infections or other complications at the time of sampling. Samples were analysed by multi-parametric flow cytometry for the expression of inhibitory receptors on T-cell subsets and the results were validated with BH-SNE, an unbiased dimensionality reduction algorithm. We exploited HLA-mimicking fluorescent molecules loaded with a specific epitope to screen anti-tumour and anti-viral T cells whereas the T-cell receptor repertoire was assessed by TRAC and TRBC RNA sequencing and the relative frequency of each T-cell receptor calculated. To evaluate T-cell function and specificity, CD107a expression, cytokine profiles and killing of autologous blasts were quantified. Results. After Haploidentical-HSCT PD-1, CTLA-4, 2B4 and Tim-3 were expressed at higher percentage when compared to HD, independently from the clinical outcome. In contrast, after HLA-matched HSCT, patients who relapsed displayed a higher frequency of BM-infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p To gain insights on the inhibited T-cell subpopulation identified in the BM of relapsing patients, we separately profiled the different T-cell memory subsets: in both HD and CR patients the IR expression was confined to effector memory and effectors whereas at relapse PD-1, 2B4, KLRG1 and Tim-3 were also expressed in BM-infiltrating central memory (TCM) and memory stem T cells (TSCM, p Interestingly, the TCR repertoire of BM-infiltrating T cells at relapse displayed a restricted clonality, suggesting that immune inhibitory signals are active on discrete and specific T-cell clones. To gain further insights on such clones, we assessed the IR expression profile on CD8 T cells specific for viral (CMV) and tumor-associated antigens (including peptides from WT1, EZH2 and PRAME). We observed a higher IR expression and co-expression on tumor-specific T cells when compared to viral-specific CD8 cells, particularly in case of patients who experienced post-transplant relapse. In accordance, IRpos sorted T cells harvested from relapsing patients showed a restricted TCR repertoire and, when challenged with autologous leukemic blasts, proved enriched in leukemic specificities as shown by higher expression of the activation marker HLA-DR (p Conclusion. These results highlight a wide, yet reversible, immunological dysfunction likely mediated by AML blasts in the BM of patients relapsing after allogeneic HSCT, that is particularly evident on memory T cells specific for tumor antigens. This suggest and open new therapeutic opportunities for AML. Figure. Figure. Disclosures Bondanza: Novartis: Employment. Vago:GENDX: Research Funding; Moderna TX: Research Funding. Bonini:Intellia Therapeutics: Research Funding.

In our previous investigation, using whole exome sequencing, we have shown that multiple myeloma (MM) patients display a complex dynamic of clonal evolution and the number of mutations in patient samples correlate with overall and relapse free survival. These results highlight the importance of understanding the mechanisms driving genomic instability in MM. Investigating mechanisms underlying genomic instability, we have shown that dysregulated homologous recombination (HR), nuclease (especially apurinic/apyrimidinic related) and APOBEC deaminase activities contribute to genomic instability in MM. We have also demonstrated that bone marrow microenvironment (BMM) also contributes to genomic instability in MM. So here we have further investigated the soluble factors in BMM that may impact genomic integrity. We treated RPMI8226 MM cells with IL-6, IL-17 and TGFb and evaluated their impact on DNA breaks by monitoring the levels of gH2AX (a DNA break marker). Treatment with all 3 cytokines (IL-6, IL-17 and TGFb) caused DNA breaks, as demonstrated by increase in gH2AX, in MM as well as a solid tumor (esophageal cancer) cell lines. Importantly, among these cytokines, the exposure to IL-6 was associated with the highest induction of gH2AX expression. These observations were confirmed in additional MM cell lines (MM1S, H929, OPM2 and U266) treated with IL-6. Since we have previously demonstrated that elevated HR is a key mechanism of genomic instability in MM, we investigated the role of IL-6 in dysregulation of HR pathway by evaluating its impact on p-RPA32 (a marker of DNA end resection which is a decisive step in the initiation of HR), recombinase (RAD51) expression and HR activity (as assessed by a functional assay). Treatment of MM cells with IL-6 led to increased expression of p-RPA32 and RAD51 (as detected by Western blotting) as well as increased HR activity in MM cells. Increased HR activity was also observed following exposure of MM cells to IL-17, TGFb and IFNb, with the highest induction caused by IL-6. However, the combination of cytokines (IL-6, IL-17 and TGFb) led to a further (> 2-fold) increase in HR activity in these cells, indicating that these soluble factors may interact in inducing mechanisms underlying genomic instability in MM. Based on our data showing important role of apurinic/apyrimidinic (AP) nuclease in regulation of HR and genome stability in MM, we also evaluated the impact of IL-6 on abasic sites, the substrate of AP nuclease activity. An increase in the number of abasic sites (ranging from 1.7- to 2.3-fold increase) was observed with increasing concentration of IL-6 in MM1S cells. To further investigated the impact of IL-6 on number of micronuclei, used as marker of genomic instability. The treatment of MM cell lines with IL-6 for 48 hrs led to a dose-dependent increase (ranging from 2- to > 2.5-fold increase) in the number of micronuclei in MM1S and RPMI cells, indicating increased genomic instability. We have previously shown that inhibition of HR by RAD51 knockdown or ABL kinase inhibitor, nilotinib, significantly reduces genomic instability, as assessed by micronuclei assay as well as direct evaluation of impact on genomewide acquisition of copy number changes over time using SNP arrays. To investigate if HR inhibition can reverse IL-6-induced genomic instability, we treated MM cells with IL-6, nilotinib and combination of both and observed that inhibition of HR by nilotinib can reverse IL-6-induced increase in number of micronuclei (by 48±17%) in MM cells. Similarly, addition of anti-IL-6 antibody also reversed the IL6-induced DNA breaks (as assessed from gH2AX expression) in MM1S cells. Moreover, combination of nilotinib and anti-IL6 antibody resulted in maximum inhibition of IL6-induced DNA breaks in MM1S cells. Taken together, these data suggest that IL-6 significantly contributes to dysregulation of HR and genome stability in MM, and agents targeting HR and/or other mechanisms of genomic instability including anti-IL-6 antibody, have potential to reduce/delay genomic evolution and disease progression. Disclosures Munshi: OncoPep: Other: Board of director.

Dysregulation of transcriptional control is a common phenomenon associated with oncogenesis. Inhibitors of DNA binding (ID) proteins are critical actors in lymphopoiesis, acting as regulators of transcription through a helix-loop-helix (HLH) domain which enables heterodimerization with basic HLH (bHLH) proteins inhibiting their binding to DNA. ID proteins have been implicated in malignant transformation, but their role in multiple myeloma (MM) is unknown. Here, we evaluated the role of ID proteins in biology and transcriptional dysregulation in MM. We first evaluated the expression of the four ID proteins in normal and malignant plasma cells using RNA sequencing data from a cohort of 360 newly diagnosed MM patients and 16 normal plasma cells. We observed significant downregulation of ID2 in primary patient MM cells in comparison to normal plasma cells (p 0.0013). To study ID2 function in MM cells, we next overexpressed ID2 in 2 MM cell lines (MM1S and NCIH929) and observed a significant decrease in proliferation rate, together with G0/G1 phase cell cycle arrest. We performed RNA-sequencing to evaluate the transcriptomic changes following ID2 overexpression. Gene set enrichment analysis (GSEA) revealed significant downregulation of genes involved in E2F pathway and significant changes in pathways related to immune response, regulation of cell death and cell proliferation. In addition, analysis of upstream cis-regulatory motifs of genes significantly dysregulated in both cell lines (>1.5 fold change) showed a highly significant enrichment for bHLH class I transcription factors (E proteins) binding motifs. Conversely, stable ID2 knockdown in 4 MM cell lines (MM1S, NCIH929, RPMI8226 and KMS11) expressing intermediate levels of ID2, showed an increased proliferation rate, assessed by cell counting, H3-thymidine incorporation and ATP production. RNA-sequencing after ID2 knockdown in MM1S and NCIH929 cells showed 600 common genes upregulated in both cell lines (>1.5 fold change). GSEA revealed upregulation of pathways involved in inflammatory response and epithelial-to-mesenchymal transition, while upstream cis regulatory motifs analysis showed a highly significant enrichment for binding motifs of bHLH class I transcription factors E proteins, in particular Tcf3 (p Next, we sought to investigate the mechanisms involved in ID2 downregulation in MM. Since the role of the microenvironment is critical in myelomagenesis, we evaluated the impact of BM microenvironment on ID2 expression in a co-culture system. Using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines, we observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 were able to significantly downregulate ID2 expression at the RNA and protein level. Furthermore, ID2 overexpression in MM cell lines (MM1S and NCIH929) abrogated the impact of BMSC on MM cell proliferation. Next, we evaluated ID2 promoter methylation profile and binding motifs using Sequenom mass array and the assay for transposase-accessible chromatin sequencing (ATAC-seq), respectively. While we didn't observe any increase in methylation of CpG islands located in ID2 promoter in co-culture, explaining ID2 downregulation, we identified several binding motifs corresponding to known driver transcription factors in MM. Especially, we identified SP1 binding motif and we confirmed SP1 binding to ID2 promoter by ChIP-sequencing in MM1S, NCIH929 and U266. These data demonstrate that in MM, ID2 acts as a tumor suppressor by promoting major transcriptomic changes and cell cycle arrest. Bone marrow stromal cells further induce significant downregulation of ID2 in myeloma cells suggesting that ID2/bHLH axis and other ID2 related pathways represent a potential new therapeutic target in myeloma. Disclosures Anderson: Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy. Munshi:OncoPep: Other: Board of director.

Besides the well described function of RNA to produce proteins, a large volume of transcribed product has non-coding function. A recent analysis of RNA repertoire has identified a family of non-coding transcripts with sequence longer than 200 nucleotides, the long intergenic non-coding RNAs (lincRNAs). Although lincRNAs have been considered to provide regulatory functions, their precise role in cellular biology remains unclear. Using our RNA-seq data from 360 newly-diagnosed patients and 18 normal plasma cells, we have recently described the landscape of lincRNAs in multiple myeloma (MM) and reported their role as independent risk predictors for survival outcome. We have now studied the functional role of a lincRNA, the miR-17-92 primary precursor linc-MIR17HG, present in our lincRNA profile and highly correlated with overall survival in MM. We observe that inhibition of linc-MIR17HG by antisense LNA GapmeRs (n=2) leads to apoptosis in 12 genotypically distinct MM cell lines as well as in 13 primary patient MM cells. These effects are not fully rescued by expression of miR-17-92 microRNAs, suggesting a distinct biological function for linc-MIR17HG in MM. We therefore performed gene expression profile in 2 MM cell lines (AMO1 and NCI-H929) and in 2 primary patient MM cells after short-term suppression of linc-MIR17HG; and, at these early time points (18-36h), we found significant downregulation of a subset of genes (FC>2; p0.4; p We next investigated whether regulation of these early targets may contribute to the activity of linc-MIR17HG. We performed a RNAi-based screening in 2 MM cells lines (AMO1 and NCI-H929) by silencing each of the linc-MIR17HG downstream target genes with at least 2 different highly-specific siRNAs. This approach revealed that silencing of acetyl-CoA carboxylase 1 (ACC1, also known as ACACA), a gene encoding the limiting enzyme in the biosynthesis of fatty acids, significantly affects MM cell growth and viability. These results were validated using stable knock-down via shRNAs, confirming ACC1 as a novel vulnerability in MM. These results provide a molecular basis for reported role of fatty acid metabolism in MM cell growth and survival. We have now evaluated two orally available inhibitors of ACC1 activity, ND-630 and ND-646, in a panel of 10 MM cell lines, an report a potent time- and dose-dependent anti-proliferative effect. The activity of these inhibitors and linc-MIR17HG on fatty acid biosynthesis in MM cells is under investigation and will be presented. We have also begun to investigate molecular pathway used by linc-MIR17HG to modulate ACC1 function. Our preliminary data suggest that linc-MIR17HG may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity. In conclusion, we highlight a transcriptional regulatory activity of a lincRNA in MM with significant functional impact that can be therapeutically translated. Disclosures Anderson: Bristol Myers Squibb: Consultancy; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

14-3-3 proteins are chaperone and scaffold proteins that exert a widespread influence on cellular processes through binding to serine/threonine-phosphorylated residues on target proteins, forcing conformational changes or influencing their interactions with other molecules. Altered 14-3-3 expression is associated with development and progression of cancer. We therefore evaluated the status of all 14-3-3 isoforms in plasma cells disorders in publically available gene expression profiling (GEP) data. Using independent patient datasets, we observed a consistent higher expression of YWHAE (coding gene for the isoform 14-3-3ε) in MM and plasma cell leukemia (PCL) patients, while no consistent differences were observed with the other isoforms. Moreover, we also confirmed higher expression of YWHAE in our RNA-seq data from 420 newly-diagnosed MM patients, with relatively low expression in normal plasma cells. Finally, 14-3-3ε was also found to be constitutively expressed at protein level in primary patient MM cells and in a large panel of MM cell lines, with significantly lower expression in healthy donor B cells. To evaluate if 14-3-3ε represents a functional dependency in MM, we performed genetic perturbation of YWHAE in a panel of MM cell lines. Depletion of YWHAE using 3 different shRNA inhibited cell proliferation and induced cell apoptosis across 5 different cell lines, independently of their genetic background. We next performed CRISPR-Cas9-mediated YWHAE knock out (KO) in H929 and JJN3 cells and observed a significant decrease in cell viability and a robust apoptotic response. H929 YWHAE KO cells infected with FLAG-YWHAE addback lentiviral construct completely rescued this phenotype, confirming that loss of YWHAE is responsible for the defective cell viability and apoptotic phenotype. These observations were corroborated by ectopic overexpression of YWHAE in H929 WT cells that significantly promoted MM cell viability. To elucidate the underlying molecular mechanisms, proteins immunocomplexed co-precipitated with FLAG in H929 KO cells with 14-3-3ε-FLAG addback were analyzed by mass spectrometry. Protein analysis revealed interaction of 14-3-3ε with a large number of proteins, enriched in mTORC1, PI3K-AKT-mTOR and unfolded protein response (UPR) pathway-related genes. Among these, TSC2 and mTORC1 proteins were further studied. WB analysis confirmed interaction of 14-3-3ε with p-mTOR (S2448) and its upstream negative regulator p-TSC2 (S939), while mTORC1 downstream targets, p-p70 S6k and p-4E-BP1, did not interact with 14-3-3ε. WB analysis also revealed activation of TSC2 and consequent inhibition of mTORC1 (via decrease of p-mTOR S2448 levels) in YWHAE KD cells. YWHAE-FLAG addback reversed these effects. Additionally, GEP data in KD cells confirmed a significant impact on mTORC1 pathways. Importantly, YWHAE expression highly correlated (R> 0.8) with genes involved in the mTORC1 pathway, including PSMC4, COPS5, EIF2S2, in our RNA-seq dataset, demonstrating a clinical significance of 14-3-3ε and mTORC1 cooperation in the context of myeloma. One of the most conserved functions of mTORC1 is to promote translation. We therefore assessed the impact of YWHAE on global translational efficiencies in MM cells, and observed significant impact on nascent protein synthesis by YWHAE modulation. 14-3-3ε KD induced 4EBP1 de-phosphorylation through inhibited mTORC1, and concomitantly induced EIF2α phosphorylation. Both effects inhibited translation initiation complex formation, mechanistically supporting a strong protein synthesis arrest. These data show the modulation of several hubs of the signaling apparatus controlling translation initiation in response to YWHAE modulation, ultimately producing a marked protein synthesis inhibition. Deregulated translational control is a central feature of MM. Our findings highlight a unique function for YWHAE as promoter of MM cell survival through regulation of mTOR-dependent protein synthesis and apoptosis. Pharmacological inhibition of YWHAE/14-3-3ε is therefore a possibility to specifically target malignancies with deregulated translational control such as MM. Disclosures Anderson: C4 Therapeutics: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

To the editor: Graft-versus-host disease (GVHD), both acute and chronic, is still a leading cause of nonrelapse mortality after transplantation.[1][1] High-dose, posttransplantation cyclophosphamide (PTCy) is an attractive approach for in vivo allodepletion across the HLA barrier in allogeneic

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only cure for several patients with high-risk hematological malignancies. Recent advances in supportive therapies have significantly reduced the treatment-related mortality over the last decades. Nevertheless graft-versus-host disease (GvHD) still represents a major complication, and research is needed to achieve further progress in this area of transplantation. Moreover, although initial studies suggested relative equivalence, recent and large prospective studies showed higher rates of acute and chronic GvHD, and worse survival in allo-HSCT from unrelated donors (URD) compared with HLA-identical sibling donors. Following our encouraging results in haplodentical setting with the more recently post-transplantation cyclophosphamide (PTCy) and sirolimus as GvHD prophylaxis (Cieri et al, 2015), in this study we investigated whether the incorporation of low dose anti-thymocyte globulin (ATG) to PTCy and sirolimus GvHD prophylactic approach, may alleviate some of the increased alloreactivity associated with URD transplants. We retrospectively evaluated a cohort of 21 haematological patients receiving URD allo-HSCT in the San Raffaele Haematology and BMT Unit, from 2013 to 2015. All subjects had high-risk hematologic malignancies, with the most common diagnosis being myeloid diseases (52%). A total of 11 patients had active disease at HSCT, while 9 were in hematologic remission. Nine patients scored high according to disease risk index defined by Armand et al, 10 scored intermediate, while only 2 had a low disease score. Myeloablative conditioning consisted of treosulfan (14 g/m2/day) on days -6 to -4, fludarabine (30 mg/m2/day) on days -6 to -2, and melphalan (70 mg/m2/day) on days -2 and -1, followed by T-replete G-CSF-mobilized PBSCs (n=19) or BM grafts (n=2). Whether 12 patients received stem cells from HLA- matched URD (fully 10/10 HLA matched), the remaining 9 had one-locus HLA-mismatched from their donors. During conditioning patients received ATG-Fresenius 5 mg/kg per day IV on day -4 to -2. In vivo B-cell depletion was obtained by rituximab (i.v. 375mg/m2 given as a single dose on day -1). Postgrafting immunosuppression consisted of PTCy (50 mg/kg/day) on days 3 and 4. Sirolimus was given orally from day 5, and monitored to maintain a target therapeutic plasma level of 5-14 ng/ml. According to chimeric status and evidence of GVHD, the dosage of sirolimus was tapered during the second month post-transplantation, ending in complete withdrawal within the sixth month after HSCT. Mycophenolate mofetil (MMF) was initiated orally at 10 mg/kg t.i.d. on day 5 after HSCT, and withdrawn on day 30. Median follow up was 247 days (range 29-486) from HSCT. All patients were engrafted in a median time of 17 days (range, 14-51 days), with full donor chimerism achieved by day 30. Poor graft function was observed in 11 patients. Post-HSCT recovery of lymphocyte subsets was lengthy as compared to other unmanipulated HSCT, with a median time to CD3>100/ml of 41 days (range, 22-389 days) by flow-cytometry, while 5 patients never reached immune reconstitution. All except one developed febrile neutropenia during aplasia. We observed a high rate of serious infections: severe pneumonia in 7 cases (5 required NIV, 4 were admitted to ICU), CMV reactivation in 13 (3 CMV disease), HHV6 reactivation in 10, BK virus haemorrhagic cystitis in 2 patients. Although the overall mortality associated with these complications was limited, as evidenced by a favourable NRM, it caused significant morbidity in patients and often required aggressive therapies, potentially contributing to poor graft function. The incidences of acute and chronic GVHD were low. A grade II-IV acute GvHD was reported in 10% patients, while five patients developed chronic GvHD (mild in 4 cases, only one moderate). Non-relapse mortality (NRM) at 100 days and 1 year was 4.8% and 20.7%, respectively. The estimated 1-year overall survival (OS) and relapse incidence were 51% and 22.5% respectively. Although limited by a small number of patients with relatively short follow-up, our study suggests that URD allo-HSCT with ATG, PTCy and sirolimus-based GvHD prophylaxis, can be very effective in preventing GvHD, but at the expense of delayed immune reconstitution and high rate of infectious complications, while warranting acceptable NRM and relapse incidence. Disclosures Bonini: MolMed S.p.A: Consultancy.

p21-activated kinases (PAKs) are effectors for the small GTPases Cdc42 and Rac that control a wide range of cellular processes including cytoskeleton reorganization, cell proliferation, survival and motility. Based on our initial results showing significant biological and functional role of PAK4 in plasma cell malignancies, we investigated its role in Waldenström macroglobulinemia (WM). We first confirmed high level of PAK4 expression and activity in WM cells. Both genetic depletion via stable lentiviral knock down and pharmacological inhibition of PAK4 via allosteric modulator KPT-9274 decreased tumor cell growth and survival in a dose and time dependent manner in a panel of WM cell lines. The growth inhibitory effect was associated with induction of apoptotic cell death, caspases activation and PARP cleavage as well as modulation of pro-apoptotic proteins such as PUMA. In addition, related-PAK4 biological processes such as migration, invasion and adhesion to bone marrow stromal cells (BMSCs) were significantly impacted by the inhibitor. A significant effect of KPT9274 was also observed in primary tumor cells from both newly diagnosed and relapse WM patients, including primary cells from ibrutinib resistant patients. To gain further insight into the consequences of PAK4 inhibition, we performed gene expression profile in WM cell lines treated with KPT-9274. Intriguingly we detected a significant impact on expression of Fanconi Anemia (FA)/BRCA pathway related genes, a DNA-damage response pathway essential for the repair of DNA interstrand crosslinks (ICLs) and regulation of cellular responses to replication stress. Since FA/BRCA deficient cells are defective in the formation of Rad51 foci, a crucial component of the homologous recombination (HR) repair machinery, we examined the effect of PAK4/NAMPT inhibition on Rad51 as well its impact on HR. We observed that, apart from inducing DNA damage, assessed by γH2AX expression, KPT-9274 specifically inhibited Rad51 and the double strand break repair by the homologous recombination pathway. Protein pull down analysis revealed the existence of a complex between nicotinamide phosphoribosyltransferase (NAMPT) and the conserved PAK binding domain; treatment with KPT-9274 disrupted this interaction and inhibited the enzymatic activity of NAMPT. NAD is necessary for PARP function especially with increasing DNA damage; and its depletion severely hamper DNA repair. Consistent with this, we observed that nicotinic acid supplementation rescued the effect of KPT-9274 on NAMPT inhibition as well as DNA damage and repair. As FA/BRCA pathway has been shown to affects sensitivity to alkylating agents, we next combined KPT-9274 with either melphalan or bendamustine in MM and WM cells. The combination treatment resulted in a synergistic time- and dose-dependent inhibition of cell survival and growth in cell lines and primary patient tumor cells. We observed a synergistic induction of apoptosis and activation of caspase-3/7, -8 and -9 following treatment with combined versus single-agent therapy. Similarly, in PAK4 knock-down WM cells bendamustine significantly increased the rate of early and late apoptotic cells (60%) compared to scrambled knockdown cells (37%). In summary, we report a significant anti-proliferative activity of dual PAK4-NAMPT inhibition in WM cells, with induction of apoptosis, DNA-damage response and FA/BRCA pathway disruption and a strong synergistic activity in combination with DNA damaging agents such as Bendamustine. Altogether, our data suggests dual PAK4-NAMPT inhibition represents a novel therapeutic strategies in WM. Disclosures Senapedis: Karyopharm Therapeutics Inc: Employment, Equity Ownership. Baloglu: Karyopharm Therapeutics Inc: Employment, Equity Ownership. Oliva: Takeda: Honoraria; Celgene: Honoraria. Anderson: Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees. Treon: Pharmacyclics: Consultancy, Research Funding.

Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.

Background: Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for many haematological diseases. Despite advances in supportive therapies, acute Graft-versus-Host Disease (aGvHD) remains the leading cause of early morbidity and mortality. There are shortcomings in the prediction of aGVHD, indicating the urgent need for non-invasive and reliable laboratory tests to allow a precision-medicine tailored prophylactic approach. Aims: We conducted a prospective observational study to ascertain the potential usefulness of the levels of eleven biomarkers, measured pre- and at +7 days post-alloHSCT, in predicting the development and severity of aGvHD in allo-HSCT patients. These time-points were chosen as early risk stratification may provide a window for additional prophylactic measures. Methods: We collected data from 120 consecutive patients (41 female and 79 male; median age 52, range 19-77 years) who underwent allo-HSCT at our institute between 2014 and 2015. Most patients were affected by myeloid malignancies (AML=52%, MDS=11%, MPN=5%) while 32% were of lymphoid origin (ALL, MM and lymphomas). Revised disease-risk index (DRI, Armand et al.) was low or intermediate for 41% of pts, high for 44% and very high for 15% of them. Most patients (n=101) received peripheral blood stem cells (PBSC). Stem cell donors were unrelated (n=39, HLA matching 9/10 in 14 and 10/10 in 25), family haploidentical (n=56), HLA-identical sibling (n=21), or cord blood (n=4). Post-transplant GvHD prophylaxis was PT-Cy-bases in 65 patients, ATG-bases in 28 patients, both agents in 18 cases whilst 9 patients received neither. Additionally, sirolimus and MMF were used as additional GvHD prophylaxis according to institutional guidelines. The following biomarkers were measured 7 days pre- and post-transplant: interleukin-6 (IL6), Ceruloplasmin(CER), Cholinesterase (CHE), Albumin, Immunoglobulin A, Gammaglutamyl-transferase (GGT), White Blood Cells, Neutrophils, Haemoglobin,Platelets and Glycaemia. ROC curves were used to define optimal cut-offs of the biochemical variables to predict 100-day severe aGvHD and 100-days TRM by logistic regression. Then cumulative incidences curves (CI) for aGvHD and TRM and overall-survival (OS) curves were computed comparing patients under and over the identified cut offs for the variables with the best reported sensitivity and specificity. Multivariate Cox proportional hazard regression was finally applied. Results: The 100-days CI of grade III-IV aGvHD was 13.5%, with a 100-days CI of TRM of 12%. The 1-year OS was 64%. Death was reported in 49 of the total 120 patients: 5/49 deaths were attributable to aGvHD. At baseline, IL6 threshold value of 2.5 pg/ml was significantly associated with the prediction of 100-days severe aGVHD (sens 65%, spec 61%) and TRM (sens 75%, spec 61%). Patients with IL-6 concentration equal or superior to 2.5 pg/ml had higher 100-days CI of severe aGvHD (16% Vs 6%, p 0,03) and TRM (12% Vs 3%, p 0,06). Interestingly, higher IL6 concentrations were associated to worse 1-year OS (42% Vs 82%, p= 129 U/l levels had higher 100-days CI of severe aGVHD (15% vs 7%, p 0,16) and TRM (13% vs 4%, p 0,01), in addition to worse OS (53% vs 71% at 1y, p 0,03). By multivariate analysis (adjusting for age, DRI, Sorror comorbidity index, type of donor, source of stem cells, and backbone of GvHD prophylaxis) pre-transplant IL6 concentrations were significantly associated to severe aGvHD (HR 3, p 0.04), TRM (HR 3, p 0.06), and OS (HR 2.6, p 0,004). Conclusion: In our series, baseline IL6 levels are predictors of aGvHD, especially with regards to grade III/IV aGvHD and translate in a higher risk of TRM and worse OS. This biomarker could be incorporated in a treatment algorithm to increase primary GvHD prophylaxis in patients at risk of severe aGVHD, potentially improving the final outcome of allo-HSCT. Disclosures Ciceri: MolMed SpA: Consultancy.

Background In an effort to reduce transplantation-related morbidity and mortality in patients (pts) undergoing total body irradiation as part of myeloablative conditioning regimen for hematological malignancies candidated to allogeneic transplant, data have been emerging in support of Helical Tomotherapy to deliver radiation doses to target organs, as bone marrow and lymph nodes, sparing normal organs. We report the first results of feasibility and efficacy of myeloablative Treosulfan-based conditioning chemotherapy combined with dose-escalating (8-10-12 Gy) TMI in pts affected by chemosensitive advanced MM (TrRaMM-TMI: Eudract N° 2013-002479-16). Patients and methods Ten pts (7 females and 3 males with a median age of 60 years), affected by chemosensitive advanced MM have been treated from December 2012 to December 2015. Most of them have received previous therapies with Bortezomib (90%) and IMIDs (80%) and all pts experienced relapse after autologous transplant; active disease was present in the 30% of the pts at the time of the transplant. The pts' median score of HCT-comorbidity index was 2. All pts received a myeloablative Treosulfan-based chemotherapy (Treosulfan 14g/mq/day on days -6 to -4; Fludarabine 30mg/mq/day on days -6 to-2), combined with TMI delivery on day -2 and -1 if 8Gy and on day -3, -2 and -1 if 10 Gy or 12 Gy, using Helical Tomotherapy. Each pt had a personalized immobilization device and before any fraction the correct set-up was controlled by a mega-voltage CT scan. Three pts have been submitted as pilot cases to TMI 8 Gy, and four and three pts respectively in the two protocol cohorts of TMI 10 Gy and 12 Gy. Peripheral-blood has been the stem cell source for all the pts: three from a matched sibling donor and seven from a matched unrelated donor (10/10 HLA match in six cases and one 9/10 HLA match). A median of 5x10e6 Cd34+/kg and of 319x10e6 Cd3+/Kg have been infused and graft versus host disease (GVHD) prophylaxis with sirolimus and mycophenolate mofetil has been administered; antithymocyte globulin on days -4 to -2 and rituximab on day -1 were added in the unrelated transplant setting. Results All pts received the planned TMI dose in each cohort; engraftment occurred in all pts and no secondary graft failure was observed. No grade 3 and 4 extra-hematological toxicities were observed, and lower grade toxicities, especially gastrointestinal, were completely reversible. No more than 10% of viral reactivations have been demonstrated and one case only of severe sepsis occurred, promptly resolved. No invasive fungal infections occured. No transplant related mortality (TRM) was registered and the two deaths were due to progressive disease. Immune reconstitution after transplant described a T-cell CD8+ and CD4+ repertoire recovery with a median number of 350/microL and of 100/microL on day +90 respectively; NK cells recovery was rapid with a median of 140/microL on day +90, while B cells were still missing on day +90. With a median follow-up time of 22 months on surviving pts, a 2-year PFS of 64% and a 2-year OS of 77% have been described. The day +100 cumulative incidence of grade II-IV acute GVHD was 40%; if grade >III 10%; chronic GVHD 1-year cumulative incidence was 42%; if moderate and severe 31%. 2-year relapse incidence was 36%. Conclusions The first data coming out from the present experience show that TMI addition to Treosulfan-based chemotherapy has not demonstrated an addition of at least short-term toxicities, confirming the combination feasibility, so that TMI 14 Gy delivery is in the plan for the third cohort of the study. Moreover the efficacy outcomes in terms of survival and acceptable TRM, suggest that myeloablative conditioning is feasible in the context of MM and especially promising even in pts with advanced and heavily pretreated disease. Disclosures Bonini: Molmed SpA: Consultancy; TxCell: Membership on an entity's Board of Directors or advisory committees. Ciceri:MolMed SpA: Consultancy.

Introduction Voriconazoleis a second-generationtriazolebroad-spectrum antifungal agent indicated in adults and children aged 2 years and above as treatment of invasive aspergillosis, treatment ofcandidaemiain non-neutropenic patients (pts), treatment of fluconazole-resistant serious invasive Candida infections, treatment of serious fungal infections caused byScedosporiumspp. and Fusarium spp. Voriconazoleis associated with a broad spectrum of dermatologically adverse reactions: it seems to be responsible for a multistep process beginning with acute and chronicphototoxicity, followed by actinic keratosis (AK), and finally skin squamous cell carcinoma (SCC), especially if therapy is maintained. Strictphotoprotectionis mandatory; drug replacement by anothertriazolemust be discussed in case of acutephototoxicity.Voriconazolemust be stopped in pts with chronicphototoxicity, and a long-term dermatologic follow-up of skin lesions is required even after withdrawal. It is now established thatvoriconazoleis an independent risk factor for the development of cutaneous malignancy in lung transplant recipients. Recently, a retrospective study from the Mayo Clinic (WojenskiDJ et al, Transplant Infectious Disease 2015, 17, 250-58) confirmed the association betweenvoriconazoleand SCC also after allo-HSCT (allogeneic hematopoietic stem cell transplantation) and identified cumulative days ofvoriconazoleas a risk factor for SCC. Methods The current study seeks to analyze the correlation betweenvoriconazoleexposure and non-melanoma skin cancer (NMSC) in our Center, where it is available an intensive dedicated follow-up after allo-HSCT to prevent and early detect second solid tumors. Results We analyze data prospectively collected at our Long-Term Follow-Up clinic between 2011 and 2016 including 302 adult pts with a minimum follow-up of 24 months. A written consent was given by pts allowing the use of medical records for research in accordance with the Declaration of Helsinki. Baseline characteristics of the 302 pts are outlined in table 1. In total, 25 pts developed NMSC - median time from allo-HSCT 42 months (range, 9 months - 20 years) - median follow-up after NMSC diagnosis 2 years (range, 2 months - 12 years). The estimated cumulative incidence of NMSC at 3 years was 3.2% and at 5 years 6.2%. At the dermatological annual evaluation 3 pts were presenting AK, only one progress to basal cell carcinoma (BCC), the 2 pts with AK are under dermatological follow-up. All pts were treated withvoriconazolefor more than 180 days. In total 19 pts were diagnosed with BCC and 6 pts with SCC. Five pts with SCC and 17 with BCC were treated withvoriconazole, overall 16/22 (4 SCC) for more than 180 days. All pts were treated according to standard practice for NMSC, unfortunately 1 pts deceased due to SCC progression. Only 2 pts were diagnosed and treated for NMSC before transplantation. Six pts had antecedent acute GvHD and 8 pts had antecedent moderate to severe chronic GvHD. History ofvoriconazoleexposure, cumulative days ofvoriconazoleuse, gender, age at transplant, TBI based conditioning regimen, acute/chronic GvHD and skin cancer pre-transplant were considered for analysis. Age at transplant above 48 years (p Conclusions Our experience confirms the correlation betweenvoriconazoleand occurrence of NMSC after allo-HSCT. Incidence of NMSC is higher than previously reported in registry reports, and the occurrence of NMSC in pts exposed tovoriconazoleseems to be precocious. This observation confirms the relevance of counseling and prevention of NMSC in patients benefiting fromvoriconazoleas a crucialmold-active antifungal prophylaxis and treatment. Disclosures Bonini: Molmed SpA: Consultancy; TxCell: Membership on an entity's Board of Directors or advisory committees. Ciceri:MolMed SpA: Consultancy.

Introduction Tyrosine kinase inhibitor (TKi) has become the standard of care in patients (pts) with chronic myeloid leukemia (CML) and an unavoidable tool in the combined therapy for pts with Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). Nevertheless, because of resistance to TKI and side-effects, allogeneic stem cell transplantation (HSCT) remains the standard therapy of ALL Ph+ and of CML pts failing 1st line therapy with TKi, with failure or insufficient response or intolerance or mutations resistant to 2nd generation TKI, or in the advanced phase at diagnosis (accelerated phase and blast crisis). Unfortunately, despite greater remission with the use of TKi pre-transplant, HSCT transplant outcome have not improved largely due to high incidence of relapse after transplant. In the past decade several multi-institutional studies confirmed the feasibility and safety of post-HSCT imatinib administration as prophylactic or therapeutic strategy. Second and 3rd generation TKi administration after HSCT - targeting mutational status and according to pre-HSCT activity - is today under investigation. Methods Here we are reporting our experience in post-HSCT treatment with the 3rd generation TKi ponatinib in 5 pts (4 CML, 1 ALL Ph+) treated between 2011 and 2016 at our Institution. Pts data and information were collected from Institutional database and chapters revision. A written consent was given by pts allowing the use of medical records for research in accordance with the Declaration of Helsinki. Results Pts and diseases features are reported in table 1. Stem cell source was peripheral blood in all cases, 3 pts were transplanted from a family mismatched donor (haplo), 1 from a family matched donor, 1 from a matched unrelated donor (MUD). The 3 haplo-transplanted pts previously underwent a MUD HSCT. All pts received a treosulfan based conditioning regimen and GvHD prophylaxis consisted on co-administration of MMF and rapamycin. Pre-transplant treatment for the ALL Ph+ consisted of chemotherapy combined with dasatinib, followed by a first MUD HSCT and dasatinib in maintenance. The patient relapsed 1 year after HSCT with documentation of mutation V299L. Ponatinib was introduced as salvage treatment to bridge second haplo HSCT. Pre-transplant treatment for the CML patients consisted of TKi therapy with combination of chemotherapy in case of uncontrolled progression of disease. Two pts received a first MUD HSCT but relapsed respectively 5 months and 4 years later. Four pts received ponatinib 45 mg daily before the last HSCT: one patient achieved sustained major molecular response, 3 pts obtained transient response. All pts were presenting 2nd generation TKi resistant mutation (ref table 1). Ponatinib was started at a median time of 157 days after HSCT (range, 117-583): in 3 cases as salvage treatment in overt relapse, while in one case as prophylaxis and one case as preemptive therapy. Acute GvHD was diagnosed in 4 pts before ponatinib administration, 2 of them also experienced chronic GvHD. No new cases of GvHD were observed after initiation of ponatinib. Immunosuppressive treatment and azoles treatment were discontinued before ponatinib in all but one patient who was under combined treatment for chronic GvHD: therapeutic drug monitoring was closely performed without evidence drug-drug interaction. Pts were regularly evaluated for toxicities. No serious adverse events were reported in our experience: we administered ponatinib at a median maximum dosage of 30 mg daily (range, 15-45 mg), for a median of 24 weeks (range, 4 - 116 weeks). Two pts required anti hypertension drugs. One patient was closely monitored for multifactorial liver cholestasis never requiring ponatinib discontinuation. At last evaluation one patient maintained the status of molecularly undetectable leukemia (follow-up post HSCT 30 months) and two pts obtained molecular response (follow-up post HSCT 25 months and 5 months). Two patients who received therapeutic ponatinib in overt relapse didn't respond and died for progressive disease. Conclusions Ponatinib is safe and well tolerated as bridge to HSCT and to maintain the disease control after transplant. Prophylaxis targeted therapy and pre-emptive therapy with ponatinib may lead the reduction of disease relapse for high-risk Ph+ leukemia. Disclosures Bonini: Molmed SpA: Consultancy; TxCell: Membership on an entity's Board of Directors or advisory committees. Ciceri:MolMed SpA: Consultancy.

Introduction Pre-transplant risk assessment is a crucial issue to improve the HCT decision-making process. Several transplant-related models have been designed to optimize decision-making about suitable candidates for allogeneic HCT. The refined Disease Risk Index (DRI) was developed to stratify disease risk acrosshistologiesand HCT regimens. However, few recipients of haploidentical HCT were originally included in the DRI study cohorts. In 2015 a first large cohort of non-myeloablativehaploidentical HCT with post-transplant cyclophosphamide (PTCy) confirmed the validity of DRI also in this setting. Beside this, in the past few years the novel composite end point of GVHD-free, relapse-free survival (GRFS) after HCT spreads out. GRFS acknowledge that both survival and rates of grade 3-4 acute GVHD, chronic GVHD requiring systemic treatment, relapse, or death are clinically meaningful. GRFS therefore represents ideal recovery from HCT and a measure of cure without ongoing morbidity. Methods We analyzed risk-stratified GRFS according to the refined DRI in haploidentical HCT at our Center, where it was exploited - since 2006 - asirolimus-based,calcineurininhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated haploidentical HCT. We analyze data collected between 2006 and 2014 including 207 adult pts. Data were prospectively collected in Institutional database. A written consent was given by pts allowing the use of medical records for research in accordance with the Declaration of Helsinki. All consecutive pts receiving a haplo HCT as 1st allogeneic transplantation were included - pts receiving haplo HCT as 2nd or 3rd HCT were excluded from the present analysis. Results Baseline characteristics of the 207 pts are outlined in table 1. With 4-year median follow-up, 4-year probabilities of transplant related mortality (TRM),relapse, progression-free survival (PFS), and overall survival (OS) were 25,8%, 42,5%, 31,7%, and 34,4%, respectively. Day-100 cumulative incidence of grade II-IV and III-IV acute graft-versus-host-disease (GvHD) were 30,1% and 15,5% respectively. The 4-year cumulative incidence of chronic GvHD was 33,5% (moderate-severe chronic GvHD 26,2%). Considering the composite end point of GRFS, for the entire population the 4-year GRFS was 17,8%. By refined DRI group, low-intermediate (n 69), high (n 105), and very high (n 33) risk groups had 4-year GRFS estimates of 31,1%, 13,7%, and 3,0% (p < .0001), with corresponding 4-year OS estimates of 56,7%, 28,9%, and 6,1% (p < .0001). On a multivariable Cox model we considered as covariates age, host/donor sex mismatch, host/donor CMV status, stem cell source, conditioning intensity, GvHD prophylaxis ATG-based versusPTCy-based, DRI stratification, HCT Comorbidity Index Score (HCT-CI). On multivariable analyses, the DRI was statistically significantly associated with GRFS, OS, PFS, relapse, TRM and grade II to IV acute GvHD (ref table 2). HCT-CI was statistically significantly associated with GRFS, OS, PFS and TRM. Conditioning intensity was associated with PFS and relapse, while GvHD prophylaxis (PTCyvs ATG) was only associated with OS. Interestingly no risk factors were clearly emerging for chronic GvHD. Conclusions The combination of a refined DRI and GRFS provide a valid tool to improve the HCT decision-making process and will help optimize patient outcomes. Disclosures Ciceri: MolMed SpA: Consultancy.

Background:Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML). Unfortunately, relapse still remains the major cause of death after HSCT. We investigated if T-cell dysfunction is associated to post-transplant relapse. Patients and Methods: To this,we longitudinally analyzed the T-cell dynamics in bone marrow (BM) and peripheral blood (PB) of 32 AML patients receiving HSCT from HLA identical (HLAid, 20 pts) or HLA haploidentical (haplo, 12 pts) donors. Samples were analysed by multi-parametric flow cytometry to investigate the expression of inhibitory receptors (IRs) on CD4 and CD8 T-cell subsets defined by CD45RA, CD62L and CD95 expression, and to assess the proportion of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Results were also analyzed with the BH-SNE algorithm, an unbiased computational method for the analysis of FACS data. To evaluate T-cell effector functions, the CD107a degranulation assay was performed and the production of cytokines (IL-2, IFNg and TNFa) was measured by intracellular staining. BM and PB were collected 60 days after HSCT and at relapse (median 237 days; 16 pts) or, when complete remission was maintained (CR; 16 pts), at 1 year. Samples from 8 healthy donors (HD) were used as controls. Results:After transplant, BM and PB T cells showed a lower CD4/CD8 ratio (p We next investigated the expression of several IRs as T-cell exhaustion markers. After haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were significantly upregulated in BM and PB T cells at all time-points, compared to HD and independently from the clinical outcome. Conversely, after HLAid-HSCT, at the late time-point, patients who relapsed, displayed a higher frequency of BM infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p To verify whether the T-cell exhaustion phenotypic profile at relapse associates with functional impairment, we evaluated T-cell effector functions upon polyclonal stimulation. Strikingly, we observed a lower degranulation ability of CD8 cells at relapse when compared to CR (p Conclusions: After HSCT, the molecular signature of exhausted CD8 cells in relapsing pts includes PD-1, CTLA-4, 2B4 and Tim-3. The expression of IRs on early differentiated central memory and memory stem T cells at relapse suggests a wide, though reversible, immunological dysfunction mediated by AML relapsing blasts. Disclosures Bondanza: TxCell: Research Funding; MolMed SpA: Research Funding; Formula Pharmaceuticals: Honoraria. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

Infections are the major cause of treatment-related morbidity and mortality in hematological patients, mostly after allogeneic hematopoietic stem cell transplantation (HSCT). Although the proportion of infection-related deaths has decreased in the past two decades, the alarming emergence of multidrug resistant (MDR) gram-negative pathogens, increasingly reported from various cancer treatment centers, represents a significant challenge. Dramatic infection-related mortality (64.4%) have been observed in allogeneic HSCT, due to carbapenem-resistant Klebsiella pneumoniae (CRKp). Timely microbiological diagnosis, active surveillance and early therapeutic strategies represent critical aspects for the management of MDR infections after allogeneic HSCT. In this study, we investigated whether a new molecular tool could be clinically relevant in the management of high-risk hematological patients, particularly in allogeneic HSCT. At San Raffaele Hematology and BMT Unit, all blood cultures positive for gram-negative bacteria in the last year, were prospectively collected and in parallel tested with Verigene® Gram-negative blood culture (BC-GN) test (Nanosphere, Northbrook, IL, USA), a microarray-based system allowing a rapid identification of genus, species, and genetic resistance determinants for a broad panel of gram-negative bacteria directly from positive blood cultures. Initially, we evaluated the reliability of the BC-GN test on 50 consecutive patients undergoing chemotherapy (n=16), autologous (n=4) or allogeneic (n=30) HSCT. The concordance with the standard blood culture was 100% considering the bacteria detectable by the system. Resistance genes (CTX-M or carbapenemases, as KPC and VIM) were detected in 15% of the isolates, and 100% were associated to the same phenotypic antibiotic resistance. We observed only 8% of phenotypic resistances not detected by the test, belonging to other kinds of resistance mechanisms not related to the genes included in the panel. Overall, Verigene BC-GN assay correctly identified 40/50 of all the gram-negative pathogens (3 Klebsiella pneumonia, 25 Escherichia coli, 5 Pseudomonas aeruginosa, 4 Acinetobacter spp, 1 Enterobacter spp, 2 Citrobacter spp), yielding a general sensitivity of 80%, which increased to 100% if only the genera and species included in the panel were considered. Then, we examined the potential clinical impact of this molecular approach in allogeneic HSCT recipients (n=30), either in an inpatient (n=24) or outpatient (n=6) management. As far as transplanted patients' care is concerned, the BC-GN test strongly influenced clinical practice. In the majority of cases we were able to early start targeted antibiotic therapy (78%), sparing or interrupting non-specific antimicrobial therapy (56%), thus reducing useless and/or potentially more toxic antibiotics and their potential impact in favouring antimicrobial resistance. Moreover, in 5/8 cases the 'triple therapy', recommended at fever onset for all CRKp-colonized patients, was spared thanks to BC-GN test results, showing the absence of resistance markers. In 95% of patients immunosuppressive prophylaxis was not reduced, thanks to a rapid control of sepsis, avoiding the risk of undesired GVHD. Early contact isolation was possible in 32% of patients, preventing the spread of infections from a patient to the others and allowing infection control. Infection-related mortality was reported only in one patient. An outpatient management was continued in 3/6 haemodynamically stable patients, avoiding unnecessary hospitalization. While conventional microbiological methods required 2-4 days, BC-GN test provided detailed results within 2 hours from blood culture positivity. The mean gain in time comparing BC-GN test to fast blood cultures was reported to be about 20 hours. In our experience, this new microarray test has provided highly accurate identification results and earlier potentially important information on antibiotic susceptibility, both confirming and excluding the presence of an MDR phenotype. The contribution to a rapid diagnosis and an earlier targeted antimicrobial therapy of Verigene BC-GN test is of high clinical relevance for the infections by MDR bacteria, a significant public health challenge worldwide, particularly in allogeneic HSCT recipients. Disclosures No relevant conflicts of interest to declare.

Introduction Haematopoietic stem cell transplantation (HSCT) is the only curative option for patients (pts) affected by high-risk haematological diseases (HRHD). However, the availability of a match donor is still an unmet-medical need. Recently alternative donor transplantations have been broadly exploited, reaching results similar to transplants from a well match donor. Anyway, whoever the donor is, the most important complication remains Graft-versus-Host Disease (GvHD). Aim of the study We evaluated incidence, characterization, stratification, treatment, treatment response and outcome at medium and long-term follow-up for both acute (a-) and chronic (c-) GvHD in haploidentical setting. We also evaluated the impact of the NIH consensus criteria on GvHD in a routine clinical application to confirm their feasibility in daily practice and not only in clinical trials use. Methods A population of 257 pts was selected from our Institutional database on the basis of their having an HRHD with indication to allogeneic HSCT and having received a HSCT from an haploidentical family donor without exclusion. HSCT were performed between January 2004 and December 2013 at our Center. No distinction between first HSCT or subsequent one was made, all consecutive haploidentical HSCT were captured. Results Time of median follow-up was 10 months; 1-year overall-survival (OS) was 46%, with better outcome for pts transplanted in complete remission (p 0.0001) confirming disease status as the leading factor for overall outcome. Transplant related mortality was estimated to be 30% at 1 year and the leading cause of death was infections. The 6-months a-GvHD incidence was 45% (119/257) - median day of presentation 21 post HSCT (range 8-89). Late-onset a-GvHD was documented in 15 pts. Grade I GvHD was documented in 33 pts (27.7%), grade II in 44 (37%), grade III-IV in 36 (30.3%) – 6 not evaluable. Skin was the most frequently involved organ (77.3%), both as single manifestation or combined. One-hundred and five pts received a first line therapy based on high-dose prednisone (2mg/Kg) and 37/105 completely abrogated the a-GvHD. At a 3-months evaluation 46% of affected pts showed complete resolution of a-GvHD, while the mortality rate was 29%. C-GvHD affected 69/257 pts and 1-year risk of onset was 25% - median day of presentation was 139 post HSCT (range 40-809) and 36/69 pts were off immunosuppressive therapy (52.3%) at presentation. According to onset presentation 53.7% were de novo, 24.6% progressive and 18.8% quiescent GvHD. Forty-one pts (59.5%) presented at declaration with overlap features. The most frequent involved organ was skin (grade I to III), as reported in 53 pts (76.8%). Skin lesions were usually accompanied with mouth lesions (35 pts – 50.7%), liver (23 pts – 33.3%) or eyes (27 pts – 39.1%) dysfunctions. The median number of involved organs was 3 (range 1-7). Mild c-GvHD was diagnosed in 10 pts (14.5%), who received topical therapy. Fifty-nine pts – diagnosed with moderate (32, 46.4%) or severe (27, 39.1%) - received a systemic treatment for c-GvHD: prednisone 1 mg/Kg alone or 0,5 mg/kg plus mTOR- or calcineurin-inhibitor for pts that were not likely to tolerate high dose steroid due to comorbidity (namely active infections, diabetes, cardiovascular diseases). At a 12-months evaluation the overall response was 48% (complete resolution 25%, partial resolution 23%), while the mortality rate was 36%. The Landmark analysis of OS at 3 months after HSCT shows that a-GvHD affected pts had a worse outcome (p= 0,068), on the contrary the Landmark analysis of OS at 18 months shows that c-GvHD did not associate with a worse outcome (p ns) but the follow up is still short. Confirming previous reports, overlap c-GvHD was related to worse survival in comparison with classic c-GvHD (p 0.0098). Conclusion Haploidentical HSCT is a valid and feasible option for pts in need of a transplant. GvHD is manageable after haploidentical HSCT, as in full matched setting. Better knowledge and insight in a/c-GvHD are providing advance in improving pts outcome. The NIH-consensus criteria are manageable in daily clinical practice and able to translate in a tailored approach to GvHD with benefit on general outcome. Further advance in the development of specific biomarkers for GvHD will provide additional crucial information for management, diagnosis and prognostication in GvHD. Figure 1 Figure 1. Disclosures Bonini: MolMed S.p.A.: Consultancy. Bordignon:MolMed: Employment.