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Strong prognostic markers able to stratify lung adenocarcinoma (ADC) patients are lacking. We evaluated whether a six-immunohistochemical markers panel (TTF1, SP-A, Napsin A, MUC5AC, CDX2 and CK5), defining the putative neoplastic “cell of origin,” allows to identify prognostic subgroups among lung ADC. We screened a large cohort of ADC specimens (2003–2013) from Torino Institutional Repository identifying: (i) marker positivity by immunohistochemistry, (ii) main morphological appearance by light microscopy, (iii) presence of “hotspot” mutations of candidate genes by Sequenom technology. To evaluate possible predictors of survival and time to recurrence, uni- and multivariable-adjusted comparisons were performed. We identified 4 different subgroups: “alveolar,” “bronchiolar,” “mixed” and “null type." Alveolar-differentiated ADC were more common in young (P =.065), female (P =.083) patients, frequently harboring EGFR-mutated (P =.003) tumors with acinar pattern (P

Background: Constitutive activation of the classical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) pathway is a clear driver of B-cell non-Hodgkin lymphomas (NHL). Bruton's Tyrosine Kinase (BTK) and Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), which lies downstream of BTK, are key mediators of the classical NF-κB signaling pathway activated by BCR and TCR receptors. JNJ-67856633 is a first-in-class MALT1 protease inhibitor. JNJ-64264681 is a BTK inhibitor with improved selectivity against BTK. Blocking the BCR pathway at multiple points using these two orally bioavailable compounds could enhance clinical activity in B-cell lymphoma patients. Methods: JNJ-67856633 and JNJ-64264681 are currently being evaluated in phase 1 clinical trials designed to establish safety, PK, PD and the Recommended Phase 2 Dose (RP2D) of each agent. Results: JNJ-67856633 is a potent, selective, orally bioavailable, allosteric inhibitor of MALT1 protease activity. The compound inhibits proliferation of activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines bearing CD79b or CARD11 mutations as well as models mimicking resistance to covalent BTK inhibitors. JNJ-67856633 exhibits potent tumor growth inhibition in two human DLBCL xenograft models, OCI-Ly3 and OCI-Ly10, and mutation selected patient derived DLBCL xenografts. Furthermore, treatment with JNJ-67856633 leads to dose dependent inhibition of the generation of Tregs (CD4+CD25+FoxP3+) following CD3/28 stimulation in vitro, suggesting a potential immune modulatory role of MALT1 inhibition. JNJ-64264681 is an orally active small molecule that is a potent, selective, and irreversible covalent BTK inhibitor. JNJ-64264681 inhibits the growth of CD79b-mutant DLBCL cell lines in vitro and potently inhibits tumor growth in xenograft- or patient-derived DLBCL models in vivo. Treatment with JNJ-64264681 and JNJ-67856633 administered together demonstrated statistically significant tumor growth inhibition compared with vehicle control in two CD79b mutant mouse lymphoma models, one based on a DLBCL cell line (OCI-Ly10) and one based on a patient-derived DLBCL model (LY2298). In both models, the combination showed increased growth inhibition compared with single agents and tumor regression in the combination arm. Synergistic anti-proliferative activity was observed in three DLBCL cell lines carrying CD79b mutations and one MCL cell line. Conclusions: Taken together, the in vitro and in vivo data for JNJ-67856633 and JNJ-64264681 suggest that combination therapy can increase the anti-tumor effect of the monotherapies and provide a more sustained response, offering strong support for clinical investigation of the combination of these two novel agents. A phase 1b combination study is scheduled to initiate. Citation Format: Ulrike Philippar, Lorena Fontan, Ivo Cornelissen, Haopeng Rui, Sriram Balasubramanian, Marcello Gaudiano, Mariette Bekkers, Luc Van Nuffel, Tianbao Lu, Tianbao Lu, John Wiener, Mark Tichenor, Tony Greway, Kathryn Packman, Bie Verbist, Yusri Elsayed, Ricardo Attar, Jacqueline Bussolari, John Gerecitano. Combination therapy of JNJ-67856633, a novel, first-in-class MALT1 protease inhibitor, and JNJ-64264681, a novel BTK inhibitor, for the treatment of B-cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1267.

Introduction: Constitutive activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NF κB) pathway is a clear driver of B-cell lymphomas, especially the aggressive activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a key mediator of the classical NF-κB signaling pathway downstream of B-cell receptor and T-cell receptor. MALT1 possesses two functions: a scaffolding function to recruit NF-κB signaling proteins and a protease function to cleave and inactivate inhibitors of the NF-κB signaling pathway. Methods: Using a high-throughput screen followed by iterative structure-activity relationship (SAR) analyses, the MALT1 inhibitor JNJ-67856633 was identified. JNJ-67856633 was evaluated using biochemical, cellular in vitro, in vivo tumor efficacy and safety models. Results: JNJ-67856633 is a potent, selective, allosteric inhibitor of MALT1 protease activity as measured by biochemical assays or downstream cellular cytokine readouts (IL6/10) or direct MALT1 substrate cleavage (RelB, BCL10). The compound inhibits proliferation of activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines bearing CD79b or CARD11 mutations as well as models mimicking resistance to covalent Bruton's tyrosine kinase (BTK) inhibitors. Furthermore, combination effects were observed in CD79b cellular ABC-DLBCL models when JNJ-67856633 was combined with a BTK inhibitor. JNJ-67856633 showed activity in organoid cultures derived from ABC-DLBCL patients. JNJ-67856633 leads to potent in vivo pharmacodynamic shutdown in CD79b- as well as CARD11-mutant ABC-DLBCL models as measured by serum IL10 or uncleaved BCL10 levels in tumors. JNJ-67856633 exhibits potent tumor growth inhibition in two human DLBCL xenograft models, OCI Ly3 and OCI Ly10. In addition, >5 patient-derived DLBCL xenografts were evaluated and activity in mutation selected models was observed. To address the role of MALT1 inhibition in T cells, primary human T cells derived from normal healthy volunteers were treated with JNJ-67856633 in vitro. Dose-dependent inhibition of the generation of Tregs (CD4+CD25+FoxP3+) following CD3/28 stimulation was observed upon treatment with JNJ-67856633, suggesting a potential immune-modulatory role of MALT1 inhibition. Conclusions: Phase 1 clinical trials assessing the safety and efficacy of JNJ-67856633 initiated in 2019. JNJ-67856633 is a combination partner for BTK inhibitors and a promising treatment option for BTKi-resistant tumors, with demonstrated preclinical activity in CARD11 mutant tumors. In addition to ABC-DLBCL, a MALT1 inhibitor is a promising treatment option for patients with CLL, MCL, WM, and FL whose tumors have been shown to be sensitive to inhibition of BTK. MALT lymphomas, characterized by MALT1 and BCL10 translocation, represent another attractive target for MALT1 inhibition. Citation Format: Ulrike Philippar, Tianbao Lu, Lorena Fontan, Nele Vloemans, Mariette Bekkers, Luc van Nuffel, Marcello Gaudiano, Katarzyna Wnuk-Lipinska, Bas-Jan Van Der Leede, Katie Amssoms, Kristof Kimpe, Bart Medaer, Tony Greway, Yann Abraham, Max Cummings, Emanuele Trella, Greet Vanhoof, Weimei Sun, Jan Willem Thuring, Peter Connolly, Haopeng Rui, Sriram Balasubramanian, John Gerecitano, Ari Melnick, Ricardo Attar. Discovery of JNJ-67856633: A novel, first-in-class MALT1 protease inhibitor for the treatment of B-cell lymphomas [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-49.

Introduction: Constitutive activation of the classical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) pathway is a clear driver of B-cell lymphomas, especially the aggressive activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a key mediator of the classical NF-κB signaling pathway downstream of B-cell receptor and T-cell receptor. MALT1 possesses 2 functions: a scaffolding function to recruit NF-κB signaling proteins and a protease function to cleave and inactivate inhibitors of the NF-κB signaling pathway. Methods: Using a high-throughput screen followed by iterative structure-activity relationship (SAR) analyses, the MALT1 inhibitor JNJ-67856633 was identified. The lead compound was evaluated using biochemical, in vitro cellular and in vivo tumor efficacy and safety models. Results: JNJ-67856633 is a potent, selective, allosteric inhibitor of MALT1 protease activity as measured by biochemical assays or downstream cellular cytokine readouts (IL 6/10) or direct MALT1 substrate cleavage (RelB, BCL10). The compound inhibits proliferation of activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines bearing CD79b or CARD11 mutations as well as models mimicking resistance to covalent Bruton's Tyrosine Kinase (BTK) inhibitors. Furthermore, combination effects were observed in CD79b cellular ABC-DLBCL models when JNJ-67856633 was combined with a BTK inhibitor. JNJ-67856633 leads to potent in vivo pharmacodynamic shutdown in CD79b- as well as CARD11-mutant ABC-DLBCL models as measured by serum IL10 or uncleaved BCL10 levels in tumors. JNJ-67856633 exhibits potent tumor growth inhibition in two human DLBCL xenograft models, OCI Ly3 and OCI Ly10, and mutation selection patient derived DLBCL xenografts. To address the role of MALT1 inhibition in T cells, primary human T cells derived from normal healthy volunteers were treated with JNJ-67856633 in vitro. Dose dependent inhibition of the generation of Tregs (CD4+CD25+FoxP3+) following CD3/28 stimulation was observed upon treatment with JNJ-67856633 suggesting a potential immune modulatory role of MALT1 inhibition. Conclusions: Phase 1 clinical trials assessing the safety and efficacy of JNJ-67856633 initiated in 2019. JNJ-67856633 is a combination partner for BTK inhibitors and a promising treatment option for BTKi-resistant tumors, with demonstrated preclinical activity in CARD11 mutant tumors. In addition to ABC-DLBCL, a MALT1 inhibitor is a promising treatment option for patients with CLL, MCL, WM, and FL whose tumors have been shown to be sensitive to inhibition of BTK. MALT lymphomas, characterized by MALT1 and BCL10 translocation, represent another attractive target for MALT1 inhibition. Citation Format: Ulrike Philippar, Tianbao Lu, Nele Vloemans, Mariette Bekkers, Luc Van Nuffel, Marcello Gaudiano, Katarzyna Wnuk-Lipinska, Bas-jan Van Der Leede, Katie Amssoms, Kristof Kimpe, Bart Medaer, Tony Greway, Yann Abraham, Max Cummings, Emanuele Trella, Greet Vanhoof, Weimei Sun, Jan Willem Thuring, Peter Connolly, Jan Linders, Haopeng Rui, Sriram Balasubramanian, Amy Johnson, John Gerecitano, Jenna Goldberg, James P. Edwards, Yusri Elsayed, Jennifer Smit, Jaqueline Bussolari, Jaqueline Bussolari, Ricardo Attar. Discovery of JNJ-67856633: A novel, first-in-class MALT1 protease inhibitor for the treatment of B cell lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5690.

JAK/STAT3 signaling pathway is often deregulated in hematopoietic disorders including peripheral T-cell lymphoma. We describe two novel mechanisms leading to the constitutive activation of STAT3 in ALK- ALCL. Oncogenic JAK1 or STAT3 mutations are associated to hyperactive pSTAT3 that regulated canonical STAT3 and ATF3 genes. Moreover, synergizing JAK1 and STAT3 mutants sustain the neoplastic growth, which can be efficiently controlled in vitro and in an ALCL patient derived tumorgraft model by JAK1/2 inhibitors. We have discovered that novel chimera, displaying concomitant transcriptional and kinase activities, are power oncogenes capable to sustain via STAT3 the ALCL phenotype and can be uniquely neutralized by a novel ROS1 inhibitor. The pharmacological inhibition of JAK/STAT3 represents a novel strategy for the treatment of molecular stratified ALCL.

Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL biology represents an urgent need. Here we report that PTCL are sensitive to transcription-targeting drugs, and, in particular, to THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7). The STAT-signalling pathway is highly vulnerable to THZ1 even in PTCL cells that carry the activating STAT3 mutation Y640F. In mutant cells, CDK7 inhibition decreases STAT3 chromatin binding and expression of highly transcribed target genes like MYC, PIM1, MCL1, CD30, IL2RA, CDC25A and IL4R. In surviving cells, THZ1 decreases the expression of STAT-regulated anti-apoptotic BH3 family members MCL1 and BCL-XL sensitizing PTCL cells to BH3 mimetic drugs. Accordingly, the combination of THZ1 and the BH3 mimetic obatoclax improves lymphoma growth control in a primary PTCL ex vivo culture and in two STAT3-mutant PTCL xenografts, delineating a potential targeted agent-based therapeutic option for these patients., T-cell lymphomas are aggressive diseases associated with poor outcome. Here, the authors show that the THZ1, a CDK7 inhibitor, suppresses STAT transcriptional activity leading to apoptosis and sensitization to BCL2 inhibitors in T-cell lymphomas.

Background Pulmonary Adenocarcinoma with Enteric Differentiation (PAED) is a rare subtype of adenocarcinoma of emerging interest, recently introduced in the 2015 WHO classification. However, little is known about major molecular signatures of this class of adenocarcinomas and information about new biomarkers totally lack. Methods We examined the NRAS, PIK3CA, EGFR, KRAS and BRAF status through mass spectrometry sequencing and ALK rearrangement by FISH in a series of 8 PAEDs. Results 1/8 (12.5%) case had a simultaneous PIK3CA mutation (E545K) and an EML4-ALK translocation. KRAS gene showed a mutation in the codon 12 in 4/8 of PAED (50%), NRAS, BRAF and EGFR genes were wild type in all tumor samples. Conclusions We concluded that PIK3CA mutations and ALK rearrangement occur also in PAEDs, while NRAS mutations might be a very rare event similarly to pulmonary adenocarcinomas of conventional type. KRAS is the prevailing gene mutated in this class of adenocarcinoma.

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.

Introduction Bio-repositories are invaluable resources to implement translational cancer research and clinical programs. They represent one of the most powerful tools for biomolecular studies of clinically annotated cohorts, but high quality samples are required to generate reliable molecular readouts and functional studies. The objective of our study was to define the impact of cancer tissue ischemia time on RNA and DNA quality, and for the generation of Patient-Derived Xenografts (PDXs). Methods One-hundred thirty-five lung cancer specimens were selected among our Institutional BioBank samples. Associations between different warm (surgical) and cold (ex-vivo) ischemia time ranges and RNA quality or PDXs engraftment rates were assessed. RNA quality was determined by RNA integrity number (RINs) values. Fresh viable tissue fragments were implanted subcutaneously in NSG mice and serially transplanted. Results RNAs with a RIN>7 were detected in 51% of the sample (70/135), with values of RIN significantly lower (OR 0.08, P = 0.01) in samples preserved for more than 3 hours before cryopreservation. Higher quality DNA samples had a concomitant high RIN. Sixty-three primary tumors (41 adenocarcinoma) were implanted with an overall engraftment rate of 33%. Both prolonged warm (>2 hours) and ex-vivo ischemia time (>10 hours) were associated to a lower engraftment rate (OR 0.09 P = 0.01 and OR 0.04 P = 0.008, respectively). Conclusion RNA quality and PDXs engraftment rate were adversely affected by prolonged ischemia times. Proper tissue collection and processing reduce failure rate. Overall, NSCLC BioBanking represents an innovative modality, which can be successfully executed in routine clinical settings, when stringent Standard Operating Procedures are adopted.

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kappa B (NF kappa B) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NF kappa B gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NF kappa B signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

The discovery of Anaplastic Lymphoma Kinase (ALK) by Stephan Morris and colleagues twenty years ago has led to an unprecedented opportunity and provided the basis for a novel and clinically powerful stratification of human cancers. The molecular and biological characterization of ALK and the recognition of alternative mechanisms of activation of the tyrosine kinase receptors have then set the basis for the development and the subsequent application of selective small molecules. These achievements have fostered a new era in oncology, and the result of this new avenue has drastically changed the expectation of many cancer patients. Here we review the mechanisms of ALK activation and the modalities that drive ALK pathogenesis.

Anaplastic large cell lymphoma (ALCL) are orphan entities representing around 20% of all Peripheral T-cell lymphoma. ALCLs can be stratified into primary cutaneous ALCL and systemic ALCL. Patients with systemic ALCL are further distinguished based on anaplastic lymphoma kinase (ALK) translocations. Unlike ALK+ patients, ALK- ALCLs are more likely to be refractory or relapse upon treatment with CHOP (Cyclophosphamide, Hydroxydaunorubicin, Oncovin, Prednisone). A small number of genetic defects and genomic translocations have been identified and associated with clinical outcome in ALK- ALCL, but cannot fully explain the poor outcomes of ALK- patients. To further probe the molecular bases of ALCL, we performed systematic epigenomic profiling in systemic ALCL through Enhanced Reduced Representation Bisulfite Sequencing (ERRBS). To investigate the methylation profile of systemic ALCLs, we first interrogated 8 ALK+ ALCL and 16 ALK- ALCL tumors. Combining methylation levels from all interrogated CpG sites, we observed overall hypermethylation at CpG-islands (CGIs; p=0.02, t-test) in ALK- tumors compared ALK+ patients but hypomethylation outside of CGIs (>10kb away from known CGIs; p=0.03, t-test). There were no significant differences on the average methylation levels at CpG-island shores, which indicate varied methylation profiles among different genomic locations. To define methylation-based signatures, we then performed supervised analysis between ALK+ and ALK- patients. We have identified 254 ALK- patients linked consistently differentially methylated promoters (>20% DNA methylation changes and FDR We then sought to investigate how methylation evolves in ALCL. We performed ERRBS of 2 primary ALCL tumors and their matched relapse samples (2 for each patient). We observed a significant hypermethylation at CGIs in relapsed samples (p=1.4e-5, paired t-test), suggesting that hypermethylation changes may be associated with tumor evolution. To test whether the hypermethylation of specific genes is linked to tumor aggressiveness, we analyzed serial passages of patient-derived tumor xenograft (PDTX) derived from primary and relapse ALCL samples. We observed consistently hypermethylation at CGIs along different implants and conserved hypermethylation of genes at the same time (p Lastly, we investigated whether a treatment with the 5-azacytidine (5-Aza) could reverse the hypermethylation profile of ALCL PDTX. Tumors from in vivo 5-Aza treated and control mice were extracted and subjected to ERRBS. As expected, we only found global hypomethylation in 5-Aza-treated samples closely mimicked methylation profiles in naive primary ALCL and early passages of PDTX. In summary, this study provides the first comprehensive characterization of methylome in ALCL. Hypermethylation is firstly associated with the aggressiveness of ALK- patients in ALCL. Also, we identified a group of hypermethylated genes associated with relapsed ALCL, a phenotype mirrored by serial passages of PDTX in vivo. As 5-Aza treatments can revert the methylome of late passages of ALCL PDTX, we speculate that this treatment may represent a novel therapeutic approach in chemo-relapsed ALCL patients. Disclosures Martin: Gilead: Consultancy, Other: travel, accommodations, expenses; Acerta: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Honoraria; Teva: Research Funding; Janssen: Consultancy, Honoraria, Other: travel, accommodations, expenses.

Introduction. T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malignancy associated with a high risk of treatment failure. Efforts to improve outcomes have focused on underlying genetic defects. However, new evidence suggests that the microenvironment can foster drug resistance/relapses. Identification of factors that contribute to microenvironment-mediated chemo-refractoriness remains an important challenge. Here, we sought to construct an in vitro platform to dissect tumor-host interactions and to optimize drug treatments using Patient-Derived Tumor Xenograft models (PDTX) of high risk adult T-ALL and engineered human endothelial cells. Methods. T-ALL PDTX were established and serially passaged in NSG mice. Engraftment was monitored by flow cytometry of peripheral blood and/or MRI. Mice were sacrificed and leukemic cells were harvested from the spleen/bone marrow. To determine the ex vivo growing conditions, we first cultured a panel of 8 "bona fide" T-ALL cell lines and 11 PDTX cells alone in complete RPMI 20% FCS supplemented with IL2, IL12, IL15 and IL7; or co-cultured with human E4-ORF1 endothelial cells (ECs) without ILs in complete RPMI 20% FCS or serum/cytokine-free media. CDK4/6, MEK, PI3K and JAK inhibitors were used at 0.1 and 1 µM alone and in combination. Cell titer glo, cell titer blue, Annexin-V and S-cell cycle analysis were used as readouts. Total RNA from cells before and after co-culture was extracted for paired-end RNA sequencing on an Illumina HiSeq2500. Results. To study the supporting role of ECs, we first co-cultured ECs with T-ALL cell lines in vitro (serum/cytokine free co-culture) and showed that ECs could reproducibly sustain the viability of 3/8 cell lines (Loucy, KOPTK1, P12 Ichikawa) serum/cytokine-free media. A partial rescue was seen with 3 additional lines (HPB-ALL, CCRF-CEM, CUTLL1), while 2 (KE37, DND41) underwent massive cell death. We next tested whether either ILs or CXCL12 could provide anti-apoptotic signals and demonstrated that KOPTK1 and Loucy were only partially rescued by IL15 or CXCL12. Conversely, IL7, although capable of inducing a robust upregulation of pSTAT5, had no effect (CCRF-CEM and CUTLL1). We then characterized 11 PDTX from 15 high-risk adult T-ALL patients. All PDTX were serially propagated and caused T-ALL in subsequent NSG mice (massive spleen and bone marrow infiltration with extensive paravertebral mass associated with paralysis and multi-organ involvement). Genomic analysis (RNA-seq) demonstrated a high concordance between primary (pre-implant) and PDTX samples. All of them were extensively studied ex vivo, demonstratingthat T-ALL PDTX cells could only survive in ILs supplemented media, even better if enriched of growth factors and supplements for the expansion of human hematopoietic cells. However, when PDTX cells were treated with targeting compounds they all underwent massive apoptosis. Conversely, individual PDTX T-ALL could be selectively rescued by ECs, allowing the construction of individual drug response profile. To extend these data, 7 PDX T-ALL samples were screened against a 430-targeted compound library in supplemented RPMI or Stem Span media. Results indicated differential cell killing and gain (NFKB, BTK) and loss (TP-53, IGF-1R) of targets. Conclusions. These data clearly demonstrate a key role of aberrantly activated vascular niche in T-ALL cell maintenance and drug resistance. We envisage that drug screening of EC+T-ALL will lead to the identification of actionable targets in each individual patient. Our report supports the potential for future personalized curative strategies aimed at targeting both tumor cells and host tissue supporting niche elements disrupting pro-tumorigenic signals within leukemia cell niches. Disclosures Foà: Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy; Pfizer: Speakers Bureau; Ariad: Speakers Bureau. Rafii:Angiocrine Bioscience: Equity Ownership, Other: Non-paid consultant.

Intro: Mature Th17 lymphocytes play a key role in the host defenses against bacteria and fungi although unchecked Th17 activation can lead to autoimmune disorders, inflammation and cancer. The engagement of CD4+ naïve T-cells via IL6 and TGFβ favors a Th17 differentiation program, which requires the presence of IL21 and IL23 to reach a complete maturation and the maintenance of Th17 phenotype. The Th17 differentiation requires multiple lineage defining transcription factors (i.e. RORα, RORγt, and STAT3) and the activation of STAT3 signaling, which regulates a plethora of factors (i.e. RORγt, IL23R), is required for the full execution of the Th17 program. We have recently shown that JAK1/STAT3 activating mutations in Anaplastic Large cell Lymphomas (ALCL) are oncogenic, but it is unknown their protumorigenic contribution in modulating/derailing the host microenviroment and the host responses. Methods: 50 FFPE ALCLs were investigated with pSTAT3, GATA3, RORγ and T-Bet antibodies using a dual color approach. CD4+ CD62L+ CD25- CD45RAhi CD4+ naïve cells of healthy donor were sorted and cultured (7 days) in KBM (IL23 (25ng/ml), IL6 (25ng/ml), IL21 (25ng/ml), IL1β (12,5ng/ml), TGFβ (5ng/ml) + 1% FBS and anti-CD3/CD28 beads (1:50). Forced expressions of WT or mutated Y640F (YF) STAT3 were achieved via lentiviral transduction. STAT3 qRTPCR, luciferase assay, ATPlite, and ELISA were used as readouts on samples collected at day 7 post-transduction. Results: IHC stains showed that pSTAT3+ ALCL preferentially co-expressed nuclear RORγ (p Conclusions: Our data demonstrated that the ectopic expression of STAT3 leads to a robust Th17 differentiation of CD4 naïve T-cells and YF STAT3 sustains their growth overtime in the presence of TCR signaling. The preferential expression of RORγ in pSTAT3+ ALCL (ALK+ and mutated STAT3 ALK-ALCL) suggest that the deregulated activation of STAT3 can regulate the plasticity of the neoplastic cells favoring the recruitment of inflammatory host cells within the neoplastic lesions. Additional studies are underway to define the lymphoma-host interactions and the role of JAK TKI in controlling the lymphoma growth and in modulating the pro-tumorigenic inflammatory phenotype of Patient Derived Tumor Xenograft Citation Format: Ramona Crescenzo, Valentina Fragliasso, Marcello Gaudiano, Marco Pizzi, Giorgio Inghirami. Somatic mutation of STAT3 leads to the preferential Th17 differentiation in human naïve CD4-positive cells and favor TCR-mediated proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2885.

Background Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer that may either arise de novo or much more commonly after hormonal therapy for prostate adenocarcinoma. Patients diagnosed with NEPC are often treated with platinum chemotherapy able to produce only short duration responses underling the urgent need of identifying novel potential therapeutic targets for this lethal disease. In the context of our Englander Institute for Precision Medicine we developed patient derived 3D NEPC tumor organoids and patient derived PDXs to test specific inhibitors on molecular targets identified by genomic analysis of native tumors. Emerging data from an integrative molecular analysis of metastatic tumors from a large cohort of castration resistant prostate cancer (CRPC) patients, including NEPC, points to a key role of the Polycomb gene EZH2 and the epigenome in the pathogenesis of NEPC. Methods Tumor organoids were developed according to protocols developed by our Englander Institute for Precision Medicine and other Institutes. Briefly the tissue biopsies (liver and bone biopsy) were washed, enzymatically digested and then seeded in a Matrigel (BD) droplet. Organoids were then characterized at both genomic (WES) and protein level (IHC) to confirm the expression of specific markers. Organoids were also subcutaneously injected in NSG mice to generate PDX for drug treatment in vivo. Results Based on the significant EZH2 overexpression in NEPC tumors by RNA-Seq and tissue microarray, we checked the expression of EZH2 and H3K273M, the readout of its activity, in NEPC organoids and we found out that both EZH2 and H3K273M were high expressed in NEPC organoids. Therefore we evaluated the effects of the EZH2 inhibitor, GSK343, in NEPC versus CRPC organoids and in the castration resistant line DU145 versus the NEPC cell line NCI-H660. We found out that GSK343 effectively inhibited H3K27me3 and resulted in a significant reduction of NEPC organoids and H660 viability while DU145 as well as CRPC organoids were insensitive to the drug. We extended our studies generating PDXs by subcutaneously injecting NEPC tumor organoids in NSG mouse. The tumor extracted from the PDXs showed a high proliferative phenotype with molecular features characteristic of NEPC as chromogranin A expression and no androgen receptor expression. NEPC PDXs were treated with the EZH2 inhibitor, GSK126, and we observed a significant reduction of tumor size along with the treatment suggesting that EZH2 is a potential therapeutic target for this highly aggressive disease. Conclusions In the Englander Institute for Precision Medicine we are generating NEPC patient tumor organoids and PDXs to unveil new targets to facilitate therapeutic decision at this late stage disease. Among the possible hits, EZH2 represents a promising drug target and a potential modulator of the NEPC phenotype. Citation Format: Loredana Puca, Wouter R. Karthaus, Dong Gao, John Wongvipat, Andrea Sboner, Marcello Gaudiano, Chantal Pauli, Rema A. Rao, Juan Miguel Mosquera, Joanna Cyrta, Theresa Y. MacDonald, Giorgio Ga Inghirami, Yu Chen, Mark A. Rubin, Himisha Beltran. Epigenetic therapy to target neuroendocrine prostate cancer using precision medicine models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3098.

Introduction. If the role of ALK chimeras is well known in the pathogenesis of ALK+ Anaplastic Large Cell Lymphoma (ALCL), the mechanisms leading to the transformation of ALK- ALCL remain elusive. Combining Whole Exome Sequencing (WES), Copy Number Variation analysis and RNAseq, we provide a comprehensive characterization of different driving genetic alterations leading to the constitutive activation of STAT3 in ALK- ALCL. Methods. The discovery panel was composed of 23 ALCL (5 ALK+ and 18 ALK). The frequency of JAK/STAT3 mutations was investigated by Sanger and deep sequencing analyses in an independent panel of 158 ALCL (88 ALK-, 26 ALK+ ALCL and 44 cALCL) and 67 PTCL. RNAseq analysis was performed in a total of 23 ALCL (18 ALK- and 5 ALK+ ALCL). Functional tests were performed by transfection/transduction of different cells lines (STAT3 -/- MEF, HEK-293T, SUPM2 TTA and SUPM2 S3S). Validation studies were executed via Western Blot, and using cells viability analyses, Luciferase Assay, 2D and soft agar colony assays. Results. WES of paired normal/tumoral DNA identified a broad number of missense mutations (n=752) with 54 genes mutated in at least two different samples, without any preferential chromosomal distribution. We focused on recurrent activating mutations in JAK1 and STAT3, known to have relevant biological activity, demonstrating that 20% of nodal ALK- ALCL and cutaneous ALCL displayed mutations in either one of them and that 38% of JAK1 or STAT3 mutants showed convergent lesions, with a single case harboring L910P JAK1, G1097D/V JAK1 and Y640F STAT3 mutations. Missense STAT3 mutations occurred within the SH2 domain (Y640F, N647I, D661Y and A662V), meanwhile recurrent JAK1 mutants targeted the kinase domain (G1097D/V). The forced expression of JAK1 mutants in MEF cells led to the constitutive activation of pSTAT3, a phenotype enhanced by co-expression of JAK1 and STAT3 mutants and linked to colony formation. The cell growth of single and double mutant MEF was efficiently controlled in vitro by JAK1/2 and Hsp90 inhibitors (ruxolitinib and PUH71). Similarly, the treatment of mice bearing a JAK1 and STAT3 mutated ALK- ALCL Patient Derived Tumorgraft (PDT) efficiently controlled the lymphoma cell growth. Next we proved that the transcriptome of reconstituted JAK1 or STAT3 mutated STAT3 MEF -/- cells showed a hyperactive STAT3 signaling pathway and the up-regulation of multiple transcription factors. Among STAT3 associated genes, we discovered ATF3 and its known transcriptional regulated genes. The relationship between pSTAT3 and ATF3 was strengthened by a Gene Set enrichment Analysis and by their down-regulation in NPM-ALK cells after genomic or pharmacological knockdowns (KD) of ALK signaling. Moreover, we showed that the protein levels of pSTAT3 and ATF3 concordantly decreased in NPM-ALK Karpas 299 cells treated with an ALK inhibitor (CEP28122) or after IL-2 starvation of NK/T-cells . The analysis of a large cohort of Peripheral T-cell lymphoma confirmed the preferential expression of “bona fide” STAT3 genes in ALK- (30%) and in ALK+ ALCL; data confirmed by immunohistochemistry approach. Because JAK1 and/or STAT3 mutations did not recapitulate all pSTAT3+ ALK- ALCL, we performed a massive parallel RNA sequencing in search of other lesions in this pathway. This approach recurrent in-frame fusions combining transcription/repressor factors (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were identified. These chimera (NFkB2/ROS1, NFkB2/TYK2, NCOR2/ROS1, PABP4/TYK2) were proven to have both transcriptional and enzymatic activities, and to be oncogenic, a phenotype that could be abrogated by a novel ROS1 inhibitor (JNJ-ROS1i-A). Lastly, we proved that NFkB2-ROS1 rescued the phenotype of NPM-ALK cells after ALK signaling ablation but not after STAT3 knock-down. Conclusions. Our data demonstrate that a subset of ALK- ALCLs displays the constitutive activation of JAK/STAT3 pathway via multiple and alternative mechanisms (single and convergent somatic mutations and translocations), which could be significantly abrogated by specific inhibitors (i.e. JAK1/2 and ROS1). The central role of STAT3 and the possibility of using novel target strategies provide new avenues in the treatment of ALK- ALCL patients, as suggested by our preclinical ALCL PDT model. Disclosures Rosenwald: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties.

Introduction The standard treatment for elderly untreated diffuse large B-cell lymphoma (DLBCL) is RCHOP21, however up to 40% of patients experienced failures. Lenalidomide showed activity in heavily pretreated DLBCL and in vivo and in vitro data demonstrated a synergism with rituximab. In the phase I trial REAL07 (Chiappella et al, Haematol 2013), FIL demonstrated that the association of LRCHOP21 was feasible in elderly untreated DLBCL and identified 15 mg lenalidomide from day 1 to day 14 as the maximum tolerated dose in combination with RCHOP21. Patients and methods. The phase II trial REAL07 was designed based on Simon's two stage design to demonstrate an improvement of overall response rate (ORR) of 15% in LRCHOP21 compared to 70% of standard RCHOP21. Secondary endpoints were progression-free survival (PFS), overall survival (OS), event-free survival (EFS) and to correlate outcome with cell of origin (COO) profile. Response was evaluated according to 2007 Cheson criteria. Inclusion criteria were: age 60-80 FIT at the comprehensive geriatric assessment; untreated CD20+ DLBCL; Ann Arbor stage II/III/IV; international prognostic index (IPI) at low-intermediate/intermediate-high/high (LI/IH/H) risk. Treatment plan was: RCHOP21 plus 15 mg lenalidomide from day 1 to 14 for 6 courses. All cases were centrally reviewed by expert pathologist; COO profile analysis was conducted with immunohistochemistry according to Hans' algorithm and with gene expression profile (DASL assay). Results. From April 2010 to May 2011, 49 patients were enrolled. Clinical characteristics were: median age 69 years (range 61-80); stage III/IV 43 (88%), IPI IH/H 30 (61%). At the end of 6 LRCHOP21, ORR was 92%. Complete remissions (CR) were 42 (86%) and partial remission 3 (6%); 3 patients (6%) did not respond and one (2%) died for homicide. At a median follow-up of 28 months, 2-year OS was 92% (95% CI: 79-97), 2-year PFS was 80% (95% CI: 64-89) and 2-years EFS was 70% (95% CI: 55-81); 2-year PFS for IPI LI was 89% (95% CI: 62-97) and for IPI IH 76% (95% CI: 47-90) and for IPI H 72% (95% CI: 36-90). Hematological and extra-hematological toxicities were mild, with no grade IV extra-hematological events and no toxic deaths during treatment. Of the 294 planned courses of LRCHOP21, 277 (94%) were administered; median dose of lenalidomide delivered was 1185 mg (94% of the planned dose); at least 90% of the planned dose of each drug was administered in 91% of the RCHOP21 courses. Median interval time between RCHOP21 courses was 21 days (range 19-48). All 49 cases underwent central pathology review and diagnosis of DLBCL was confirmed. Regarding COO analysis, tissue block or stained slides were collected in 40/49 (82%), of which 32 were adequate for analysis. At the time of this abstract, COO analysis was reported according to immunohistochemistry data; DASL analysis is ongoing. Clinical characteristics between germinal center (GCB, 16 patients) and non-GCB (16 patients) were superimposable, excepted for a majority of H IPI risk in non-GCB group (p 0.067). ORR for GCB and non-GCB were 88% (CR 81%) and 88% (CR 88%), respectively. At a median follow-up of 28 months, 2-year PFS was 71% (95% CI: 40-88) in GCB-group and 2-years PFS was 81% (95% CI: 51-93) in non-GCB-group (Figure 1). Conclusions. In conclusion, LRCHOP21 is effective, also in poor risk patients, namely in non-GCB subgroup. These encouraging data warrant a future phase III randomized trial comparing LRCHOP21 vs. RCHOP21 in untreated non-GCB DLBCL. Disclosures: Off Label Use: lenalidomide in first line DLBCL is off lable. drug provided free by Celgene. Vitolo:Roche: Speakers Bureau; Celgene: Speakers Bureau; Takeda: Speakers Bureau.