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We aimed to assess HKII expression and its prognostic significance in diffuse large B-cell lymphoma (DLBCL) patients. The HKII protein level was determined by immunohistochemistry in 159 newly diagnosed DLBCL patients, and its relationship with overall response rate, progression-free survival (PFS), and overall survival (OS) was analyzed. HKII was expressed in 95 DLBCL patients (59.7%). HKII-positive patients had poorer outcomes than negative patients for 5-y PFS (68% vs. 84%, p = 0.029) and 5-y OS (78% vs. 94%, p = 0.05). When only patients without no bulky disease, B symptoms, or extranodal involvement who had low IPI scores were considered, those with positive HKII had worse 5y-PFS and 5y-OS (p 0.05). Multivariate analysis indicated that HKII status was an independent prognostic factor of OS. In subgroup analysis, HKII expression was associated with inferior OS in the CHOP group (p = 0.017). In CHOP group patients without bulky disease or extranodal involvement who had low LDH and low IPI scores (p 0.05), positive HKII was associated with worse PFS and OS. No differences in PFS and OS, or any independent prognostic factors, were found in the RCHOP group. In DLBCL, HKII is valuable as a prognostic biomarker and may be useful as a tool for assessing disease risk.

Coronavirus disease 2019 (COVID-19) infection has been linked to numerous autoimmune manifestations. Neither the mechanism nor the etiology of this association has been fully explored or elucidated. Prior studies have detected myositis in patients with proven COVID-19 infection, suggesting a relationship between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the development of myositis. Studies have reported elevated levels of autoimmune antibodies, including myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs), in patients with COVID-19 infection, however the prevalence is not well documented. Our objective was to assess the prevalence of MSAs and MAAs in COVID-19 patients compared with unaffected subjects. Serum samples from 74 unvaccinated, polymerase chain reaction (PCR)-positive COVID-19 infected patients were compared with serum samples from 41 healthy, unaffected individuals. All serum samples were tested for MSA and MAA reactivity. Within the COVID-19-positive group, six (8.1%) patients exhibited MSA/MAA positivity, compared with only one (2.4%) individual from the control group. Although a higher prevalence of MSA/MAA positivity was observed within the COVID-19 infected group, the difference did not reach statistical significance (p=0.223). The autoantibodies detected in this study have a unique association with dermatomyositis and other inflammatory myopathies, and may play a role in COVID-19-associated myopathy. This article was previously presented as an abstract at Jacobs School of Medicine and Biomedical Sciences Research Day on June 3rd, 2022.

Following a similar report on multiple myeloma, Ghione and colleagues report the expected observation that patients with non-Hodgkin lymphoma (NHL) receiving anti-B cell therapies have markedly reduced antibody responses to COVID-19 immunization. Although there is no information regarding T-cell immunity, this suggests that while vaccination is certainly still recommended for this population, patients should be strongly encouraged to maintain social distancing precautions and should be revaccinated after an appropriate interval from the end of their antilymphoma therapy.

Purpose: Developing novel therapy strategies to treat relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) is critically needed. Previous studies in our group found despite dysregulation of apoptotic pathway in resistant DLBCL, upregulation in glycose metabolism and energetic ATP production were also found in r/r DLBCL. Therefore, decreasing or blockage ATP production may provide a novel method to overcome resistance in lymphoma. ATP/ADP is hydrolyzed into AMP by NTPDases, and AMP into adenosine and phosphate by ecto-5’-nucleotidase, known as CD73. CD73 is the rate limiting enzyme anchored the surface of tumor and immune cell. The expression level of CD73 on the tumor was reported significantly associated with inferior prognosis in variety of cancers. Moreover, expression level of CD73 on immune cell has been shown immunosuppression in multiple cancers. Targeting CD73 by monoclonal antibody or small inhibitor currently underwent multiple clinical trials. However, the role of CD73 in r/r DLBCL and the relationship between CD73 and CD20 have not been explored. Methods: Germinal center B-cell like, activated B-cell like lymphoma, Burkitt’s lymphoma and rituximab resistant cell lines generated in our lab were used. Surface expression of CD73 and CD20 were determined by staining anti-CD73 FITC and anti-CD20 Per-cy5.5 antibodies and running flow cytometry. The expression levels of CD73 and CD20 were detected by media fluorescence index (MFI). The CD20/CD73 ratio was calculated by CD73 MFI/CD20 MFI. Result: Previously our group found surface CD20 levels exhibited a linear correlation with rituximab induced cellular mediated cytotoxicity. The CD20 MFI of germinal center B-cell like, activated B-cell like lymphoma, Burkitt’s lymphoma and rituximab resistant cell lines were: DHL4 8652, DOHH2 6472, TMD8 3981, RL 2849, Raji 2011, U2932 604, RL 4RH 599 and Raji 4RH 576. The higher MFI of CD20, the more sensitive to rituximab treatment. Here we discovered that the surface level of CD73 had an inverse correlation with CD20 level in these cell lines. In detail, CD73 MFI were: DHL4 464, DOHH2 387, TMD8 363, RL 299, Raji 611, U2932 486, RL 4RH 631 and Raji 4RH 702. The CD20/CD73 ratio of cell lines from high to low were DOHH2, DHL4, TMD8, RL, Raji, U2932, Raji 4RH and RL 4RH, respectively. This CD20/CD73 ratio in each of cell lines was found more straight forward to represent the sensitivity of rituximab or chemo drug treatment in vitro condition. Conclusion: Our study was first time to reveal the association between CD20/CD73 ratio and immune-chemo response in lymphoma cell lines, especially in resistant lymphoma in vitro condition. This may give us prognostic value of CD20/CD73 ratio in predicting lymphoma response to immune/chemotherapy in clinical setting. Further study to find CD20 and CD73 association in the clinical setting and mechanism of action need to be warranted. Citation Format: Juan J. Gu, Cory Mavis, Jessica Wang, Taylor Mandeville, Francisco Hernandez-Ilizaliturri. Prognostic value of CD20/CD73 ratio in sensitive and relapsed/refractory diffuse large B-cell lymphoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1751.

Intercellular adhesion molecule-1 (ICAM-1) is a cell-surface receptor contributing to lymphocyte homing, adhesion and activation. The prognostic significance of the protein is unknown in diffuse large B-cell lymphoma (DLBCL) in post-rituximab era. We detected expression of ICAM-1 immunohistochemically in 102 DLBCL tissue samples. Overexpression of ICAM-1 was found in 28 (27.5%) cases. In patients with low ICAM-1 expression levels, the addition of rituximab to CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy resulted in an improved overall response rate, progression-free survival (PFS) and overall survival (OS) (P=0.019, 0.01, 0.02). In pre-clinical models, we found that chronic exposure of cell lines to rituximab led to downregulation of ICAM-1 and acquirement of a rituximab resistant phenotype. In vitro exposure of rituximab resulted in rapid aggregation of B-cells regardless of the ICAM-1 expression levels. MTT assay showed knockdown of ICAM-1 could cause rituximab resistance. Neutralization of ICAM-1 did not affect rituximab activity in vitro and in vivo. Our data illustrated that in post-rituximab era, R-CHOP significantly improved the ORR, PFS and OS in ICAM-1 negative subset patients. Downregulation of ICAM-1 may contribute to rituximab resistance, and that rituximab, by promoting cell-cell aggregation, may sensitize cells to the cytotoxic effects of chemotherapy agents.

The patients with relapsed and refractory diffuse large B-cell lymphoma (DLBCL) have poor prognosis, and a novel and effective therapeutic strategy for these patients is urgently needed. Although ubiquitin-specific protease 1 (USP1) plays a key role in cancer, the carcinogenic effect of USP1 in B-cell lymphoma remains elusive. Here we found that USP1 is highly expressed in DLBCL patients, and high expression of USP1 predicts poor prognosis. Knocking down USP1 or a specific inhibitor of USP1, pimozide, induced cell growth inhibition, cell cycle arrest and autophagy in DLBCL cells. Targeting USP1 by shRNA or pimozide significantly reduced tumor burden of a mouse model established with engraftment of rituximab/chemotherapy resistant DLBCL cells. Pimozide significantly retarded the growth of lymphoma in a DLBCL patient-derived xenograft (PDX) model. USP1 directly interacted with MAX, a MYC binding protein, and maintained the stability of MAX through deubiquitination, which promoted the transcription of MYC target genes. Moreover, pimozide showed a synergetic effect with etoposide, a chemotherapy drug, in cell and mouse models of rituximab/chemotherapy resistant DLBCL. Our study highlights the critical role of USP1 in the rituximab/chemotherapy resistance of DLBCL through deubiquitylating MAX, and provides a novel therapeutic strategy for rituximab/chemotherapy resistant DLBCL.

Background R-CHOP with or without radiotherapy is the standard treatment for limited-stage diffuse large B-cell lymphoma (DLBCL). To prevent overtreatment, we assessed whether four cycles of CHOP plus six applications of rituximab was adequate with negative interim PET/CT and the role of consolidation radiotherapy specifically for patients with Waldeyer’s ring DLBCL. One hundred and twenty-nine patients with limited-stage DLBCL were enrolled in this open-label, nonrandomized, single-arm, phase 2 clinical trial (NCT01804127). Methods All patients were initially treated with 4 cycles of R-CHOP and underwent interim PET/CT. Patients with negative PET/CT (Deauville scores 1–2) received 2 additional cycles of rituximab monotherapy, unless they had any risk factors (primary mediastinal large B-cell lymphoma, extranodal primary or bulky disease). Otherwise, patients received another 2 cycles of R-CHOP. Patients with partial response on interim PET/CT received another 4 cycles of R-CHOP. No radiotherapy was conducted in Waldeyer’s ring DLBCL patients with negative PET/CT. The primary endpoint was 3-year progression-free survival (PFS). Overall survival (OS) in this study was compared with those from a historical study (NCT 00854568159). Results One hundred fifteen interim PET/CT scans (89.1%) were negative after 4 cycles of R-CHOP. An elevated lactate dehydrogenase level was significantly associated with positive interim PET/CT (P P = 0.24) and 3-year OS (85.7% vs. 95.6%, P = 0.16). There were no PFS or OS differences found between patients treated with 4R-CHOP+2R and those treated with 6R-CHOP from a historical control study. Patients with Waldeyer’s ring DLBCL and negative interim PET/CT achieved a 3-year PFS of 87.2% and a 3-year OS of 89.7%. Conclusions Our results suggested that for interim PET/CT-negative patients without risk factors, the extra 2 cycles of CHOP might be omitted, and radiotherapy might also be omitted in patients with Waldeyer’s ring DLBCL without compromising the efficacy. These results need to be confirmed in a randomized study. Trial registration clinicaltrials.gov, NCT 01804127. Date of first registration: 05/03/2013.

Background: Targeting programmed cell death-1 (PD-1) or programmed cell death ligand-1 (PD-L1) has been shown improved clinical efficacy in a wide range of tumor types. We evaluated serum interleukin 14α (IL14α) as a biomarker to predict the response of anti- PD-1 therapy. Patients and methods: Thirty advanced cancer patients treated with PD-1 inhibitor were enrolled in this study. Serum levels of IL-14α were tested at baseline and after 2 cycles of treatment. Result: Among these 30 patients, the mean expression level of IL14α before treatment was 2.1±1.21, whereas the mean level of IL14α after 2 cycles was 1.99±0.82. There were no association between the expression levels of IL14α and clinical outcome. Early change of IL14α after 2-cycles of anti-PD-1 therapy was calculated as delta IL14α % change = (IL14α level after 2 cycles - IL14α level before treatment)/IL14α level before treatment*100%. Receiver operating characteristic (ROC) was analyzed to get a cutoff point of delta IL14α % change as 2.46% (Sensitivity 85.71%, Specificity 62.5%; AUC=0.7277, P=0.034). Using this cutoff to subgroup the patients, we found higher delta IL14α % change was significantly associated with superior overall response to anit-PD-1 therapy (p=0.0072) and had a better progression free survival (p=0.0039). Conclusion: Early changes of serum IL14α level may be a useful predicting factor in advanced cancer patients with anti-PD-1 therapy. Keywords: IL14α, serum biomarker, anti-PD-1 therapy response, cancer Citation Format: Bu Hai Wang, Cai Yue Chen, Xian Zhang, Yu Xiang Huang, Yi Chun Zeng, Lei Li, Mao Qi Wang, Jing Liang Guo, Qiu Xian Li, Long Shen, Juan J. Gu, Yi Chen Liang. The early change of serum interleukin 14α levels predicts the response to anti-PD-1 therapy in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5091.

Introduction: T-cell lymphoma (TCL) accounts for 15% of all non-Hodgkin’s lymphoma cases with most patients developing relapsed/refractory (R/R) disease. The management of R/R TCL is challenging and responses to second and beyond-line therapy are generally poor. Pre-clinical and clinical studies have identified the relevance of PI3K/AKT/mTOR signaling pathway in TCL biology. The combination of PI3K inhibitor (PI3Ki) Duvelisib and the HDACi Romidepsin improved the overall response rate (ORR) of R/R TCL to 50-55% in a phase I/II study (Horwitz et. al. ASH 2018, Lugano 2021). Evaluation of PI3Ki and HDACi combinations in TCL pre-clinical models is needed to guide the design of future clinical trials. Methods: A panel of TCL cells lines were chosen representing T-cell acute lymphoblastic leukemia, T-lymphoblastic lymphoma, cutaneous T-cell lymphoma. A panel of PI3Ki (Omipalisib, Umbralisib and Duvelisib) and HDACi (Entinostat and Romidepsin) were selected to determine which combination would be most synergistic. Entinostat, an oral HDACi, was chosen for its remarkable preclinical and clinical activity in other lymphoma subtypes. Umbralisib is a new PI3Kδ inhibitor which was recently approved for treatment of follicular lymphoma and marginal zone lymphoma. Cells were exposed to Omipalisib (3.125-100nM), Umbralisib (1-320uM), or Duvelisib (1-100uM) alone or in combination with Entinostat (0.08-10uM) or Romidepsin (0.25-6) for 24, 48 and 72 hrs. Cell viability was measured using the CellTiter-Glo viability assay. Half-maximal inhibitory concentration (IC50) at 72hrs was determined for each drug and coefficient of synergy was calculated using Calcusyn Software. Changes in cellular metabolism and cell cycle distribution after exposure to PI3Ki were determined using DiOC6 staining and Propidium Iodide staining respectively. Epigenetic changes following exposure to Entinostat or Romidepsin were investigated for possible mechanisms. Results: In vitro exposure of TCL cell lines to PI3Ki or HDACi resulted in dose-dependent cell death. The IC50 at 72 hrs was 30-500nM for Omipalisib, 9-35uM for Umbralisib, 1-35uM for Duvelisib, 0.2-2uM for Entinostat and for 1-4nM Romidepsin. All HDACi and Pi3Ki combinations demonstrated additive and/or synergistic activity. Duvelisib combined with Entinostat or Romidepsin exhibited the most synergy. Methylation profiling demonstrated genomic wide hypomethylation, specifically DNMT3A, in Romidepsin treated samples compared to Entinostat. Conclusions: PI3Ki and HDACi combinations show significant synergy in TCL preclinical models. DNMT3A hypomethylation following Romidepsin exposure is being explored as the basis of PI3Ki and HDACi synergy. If confirmed in clinical trials, these results may refine the tolerability of the regimen for patients treated with this very promising PI3Ki/HDACi combination. Citation Format: Gabrielle Hartman, Cory Mavis, Juan J. Gu, Amber White, Taylor Mandeville, Pallawi Torka, Paola Ghione, Francisco J. Hernandez-Ilizaliturri. Novel pharmacological approaches of inhibiting the PI3K/AKT/mTOR signaling pathway in T-cell lymphoma (TCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2661.

Clinical observations suggest the existence of shared resistance pathways between rituximab and chemotherapy agents. To explore the mechanisms of rituximab resistance, our group created rituximab-resistant cell lines (RRCLs), which display altered expression of several inhibitor of apoptosis (IAP) family proteins. Here, we provide evidence to support pharmacologically targeting IAPs in lymphoma with LCL-161, a small molecule mimetic of the second mitochondria-derived activator of caspases (SMAC). The antitumor effect of LCL-161 was determined using luminescent adenosine triphosphate assays, flow cytometry, SCID mouse xenografts, and ex vivo patient biopsy sample studies. In vitro exposure to LCL-161 also resulted in a dose-dependent decrease in IAP levels, along with synergistic enhancement of the antitumor effect of cytotoxic chemotherapy, in rituximab-sensitive cell lines and RRCLs. In addition, LCL-161 increased the cytotoxic effect of the proteasome inhibitor carfilzomib in ex vivo lymphoma patient samples. The combination of LCL-161 with the chemotherapy regimen rituximab, gemcitabine, and vinorelbine (RGV) improved in vivo survival compared with RGV alone in severe combined immunodeficient mice implanted with RRCLs but not in animals implanted with rituximab-sensitive cell lines. In summary, LCL-161 exhibits synergistic antitumor activity in both in vitro and in vivo models of resistant lymphoma. Our data support further preclinical investigation of LCL-161 as a novel antilymphoma agent.

Rituximab/chemotherapy relapsed and refractory B cell lymphoma patients have a poor overall prognosis, and it is urgent to develop novel drugs for improving the therapy outcomes. Here, we examined the therapeutic effects of chidamide, a new histone deacetylase (HDAC) inhibitor, on the cell and mouse models of rituximab/chemotherapy resistant B-cell lymphoma. In Raji-4RH/RL-4RH cells, the rituximab/chemotherapy resistant B-cell lymphoma cell lines (RRCL), chidamide treatment induced growth inhibition and G0/G1 cell cycle arrest. The primary B-cell lymphoma cells from Rituximab/chemotherapy relapsed patients were sensitive to chidamide. Interestingly, chidamide triggered the cell death with the activation of autophagy in RRCLs, likely due to the lack of the pro-apoptotic proteins. Based on the RNA-seq and chromatin immunoprecipitation (ChIP) analysis, we identified BTG1 and FOXO1 as chidamide target genes, which control the autophagy and the cell cycle, respectively. Moreover, the combination of chidamide with the chemotherapy drug cisplatin increased growth inhibition on the RRCL in a synergistic manner, and significantly reduced the tumor burden of a mouse lymphoma model established with engraftment of RRCL. Taken together, these results provide a theoretic and mechanistic basis for further evaluation of the chidamide-based treatment in rituximab/chemotherapy relapsed and refractory B-cell lymphoma patients.

This case study reviews the design and development of a mobile-based intelligent pain management system (IPMS) app in cancer patient care and pain management in a rural hospital in China. Healthcare professionals were involved throughout the design to the evaluation stages. The IPMS facilitated real-time pain recording and timely intervention among cancer patients with pain. To evaluate the effectiveness of the IPMS, a clinical trial was administrated under the supervision of healthcare professionals. The result confirmed that the IPMS was a feasible, effective, and low-cost pain management tool for cancer patients and healthcare professionals. This case provides preliminary data to support the potentials of using IPMS in cancer pain management and emphasized that the involvement of healthcare professional throughout the system development lifecycle is crucial to the successful implementation of the IPMS.

Purpose: The purpose of this study was to investigate the effect of MYC and TP53 comutations on the clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in Chinese patients with advanced EGFR-positive nonsmall-cell lung cancer (NSCLC). Patients and methods: Tissue samples and information from 65 patients with advanced NSCLC in Northern Jiangsu People's Hospital were collected and analyzed by next-generation sequencing (NGS). Progression-free survival (PFS) and total survival (OS) were the main endpoints, and the objective response rate (ORR) and disease control rate (DCR) were the secondary endpoints. Result: Among 65 patients, 17 had TP53 and MYC wild-type mutations ( WT/WT), 36 had TP53 mutant and MYC wild-type mutations ( TP53/WT), and 12 had coexisting MYC/TP53 mutations ( MYC/TP53). When 12 patients with MYC/TP53 comutation were compared with the other two groups ( TP53/WT, WT/WT), mPFS and mOS are significantly lower than those in the other two groups (mPFS: 4.1 months vs 6.0 months, 12.3 months, HR: 0.769, 95% CI: 4.592-7.608, P = .047. mOS: 14.6 months vs 24.1 months, 31.5 months, HR: 3.170, 95% CI: 18.786-31.214, P < .001), and the ORR, DCR of patients with MYC/TP53 comutation was lower than that of the other two groups (ORR, 25% vs 44.4%, 70.6%, P = .045. DCR, 58.3% vs 72.2%, 82.4%, P = .365). Conclusion: Patients with MYC/TP53 comutations with EGFR-positive advanced NSCLC are more likely to develop drug resistance after early treatment with EGFR-TKIs and have a worse clinical outcome.

In order to identify cellular pathways associated with therapy-resistant aggressive lymphoma, we generated rituximab-resistant cell lines (RRCL) and found that the acquirement of rituximab resistance was associated with a deregulation in glucose metabolism and an increase in the apoptotic threshold leading to chemotherapy resistance. Hexokinase II (HKII), the predominant isoform overexpressed in cancer cells, has dual functions of promoting glycolysis as well as inhibiting mitochondrial-mediated apoptosis. We found that RRCL demonstrated higher HKII levels. Targeting HKII resulted in decreased mitochondrial membrane potential, ATP production, cell viability; and re-sensitization to chemotherapy agents. Analyzed gene expression profiling data from diffuse large B-cell lymphoma patients, high-HKII levels were associated with a shorter progression free survival (PFS) and/or overall survival (OS). Our data suggest that over-expression of HKII is associated with resistance to rituximab and chemotherapy agents in aggressive lymphoma and identifies this enzyme isoform as a potential therapeutic target.

Background: Extranodal Natural killer (NK) /T-cell lymphoma, nasal type (NNKTL), a unique and aggressive neoplasm, is commonly observed in East Asia and Latin America. Even at early stages, NNKTL has a poor clinical outcome because intrinsic chemotherapy resistance, rapid local disease progression and distant metastasis. Despite the use of high dose radiation therapy with or without multiagent chemotherapy regimens, NNKTL has a high rate of relapse (25-40%). Moreover, the estimated 5-year survival on relapsed NNKTL is 52.3%. The identification and validation of readily available biomarkers that predict treatment outcomes can aid in the use of consolidation cellular therapy (i.e. allogeneic bone marrow transplant) and tailored surveillance programs. Elevated serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) had been associated with inferior outcomes in non-Hodgkin lymphoma, but their utility in NNKTL remains unidentified. Here, we examined sICAM-1 levels in patients of early-stage NNKTL and its correlation with clinical characteristics, response to the therapy and clinical outcomes. Material and Methods: This was a retrospective observation study conducted in patients diagnosed as Stage I/II NNKTL in the upper aerodigestive tract treated at Shanghai Cancer Hospital, Shanghai, China and for which biospecimens collected at diagnosis and at the end of therapy (EOT) (frozen serum) were available. The study was approved by the IRB (Protocol number). Baseline and EOT sICAM-1 levels were measured by ELISA (R&D systems, Minneapolis, MN, sensitivity =0.35ng/ml) according to the manufacture' s protocol. Differences of sICAM-1 expression between baseline and EOT were analyzed by paired t-test. Difference of sICAM-1 between complete response and non-complete response groups were determined by Mann-Whitney U-test. Comparing sICAM-1 among different clinical characteristics was determined by Chi square and Fisher's test. Differences in overall survival (OS) and progression-free survival (PFS) between patients with high and low sICAM-1 levels were analyzed by the Kaplan-Meier method. Results: A total of 74 patients were included in the study (18F/56M), the median age of the patients was 45.5 (19-73). Patients were treated with radiation therapy alone (N=34), or combined modality therapy (N=40). As previously noted in other studies, achievement of a complete response at the end of therapy was associated with a better PFS and OS (P The net change (EOT minus Baseline) of sICAM-1 levels (ΔsICAM-1) was significantly higher is patients with relapse disease (81.4+63.1 vs 33.5+81.2, p Conclusion: Higher sICAM-1 levels at diagnosis were associated with primary treatment failure in early stage NNKTL. In addition, an elevated ΔsICAM-1 in patients achieving a complete response to first like therapy were associated with relapse disease and shorter PFS. Together, our data suggests that sICAM-1 may be used as serological biomarker to monitor and predict clinical outcome of early stage NNKTL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Patients (pts) with cancer have increased morbidity and mortality associated with the development of SARS-Co-V2 infection. The mRNA vaccines BNT162b2 and mRNA-1273 have robust safety and efficacy with 95-97% prevention of severe COVID-19 disease and development of protective antibody titers in 92 - 100% of healthy individuals. By contrast, some pts with hematological malignancy fail to produce anti-spike antibodies (Ab) despite full courses of vaccination. This is particularly true for pts with non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL) who are actively treated with or have received B cell-directed therapies (BCT). We recently demonstrated that NHL pts who received the COVID-19 vaccine within 9 months from BCT demonstrated markedly lower rates of seroconversion (11%) compared to healthy individuals (100%) or a cohort of older (age >65y) residents of a nursing home (91.5%). NHL pts who had received BCT more than 9 months before the vaccine responded more robustly (88%) (Ghione et al, Blood 2021). Here, we update the results of our earlier study and perform an analysis to identify factors that may help predict for adequate response to COVID-19 vaccines. We hypothesized that neutrophil (N) or lymphocyte (L) counts and/or N/L ratio (NLR) at baseline might predict for adequate Ab production in response to COVID-19 vaccines after receipt of BCT. Methods: This was an observational study performed at Roswell Park Comprehensive Cancer Center. Pts with NHL/CLL who had received COVID-19 mRNA vaccine were included, vaccine response was assessed as previously described (Ghione et al, Blood 2021). For NLR calculation, pts with CLL or NHL with blood involvement were excluded. Clinical variables were analyzed with the t test and Fisher's exact test; validity and cut-off for the L count was obtained with the ROC analysis using GraphPad9 and SPSS. Results: A total of 142 pts with various types of NHL and CLL receiving standard of care treatments were enrolled. Five pts with prior exposure to COVID-19 infection were excluded from the analysis, reaching a total n=137. Of 83 pts with NHL (n=57) and CLL(n=26) in our cohort who were vaccinated within 9 months of BCT, 14 (17%) seroconverted. Baseline N count (p= 0.5), sex (p= 0.2), age (p= 0.8), number of prior lines of treatment (p= 0.4), type of disease (p= 0.7), time from end of BCT to vaccine (p= 0.7) and type of vaccine (p= 0.08), did not affect the rate of seroconversion. For analysis of N and L counts, 37/137 pts with CLL/NHL involving peripheral blood were excluded. Among the remaining 100 pts, 76 had received/were receiving BCT and 24 were either on observation or were on a treatment not including BCT. Only 26/76 (34%) pts on treatment/previously treated with BCT mounted IgG Ab response, while 22/24 (91.6%) patients who were not on BCT mounted the IgG Ab response (p= 0.007). In these NHL pts (N=100), higher L counts and higher NLR were associated with an increased IgG response to the vaccine (p= 0.019). For pts on BCT (N=76) a higher L count (cut-off of 1455 lymphocytes per cubic millimeter of blood [µL] as noted in the ROC) was associated with a higher rate of COVID-19 vaccine response (p= 0.020, with a sensitivity of 84% and specificity 71.5%). Conclusion: In this study, we confirm that pts with NHL/CLL receiving COVID-19 vaccination while on active treatment or within 9 months of treatment with BCT respond poorly to COVID-19 mRNA vaccination (17% seroconversion). Although a higher lymphocyte count and NLR ratio were associated with improved seroconversion rates, these were not powerful predictors. Further study of specific lymphocyte sub-populations that contribute to effective vaccine induced immunity in the context of BCT is ongoing. These studies will help to define optimal strategies for immunization in patients receiving BCT. Figure 1 Figure 1. Disclosures Torka: TG Therapeutics: Membership on an entity's Board of Directors or advisory committees. Griffiths: Takeda Oncology: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Research Funding; Abbvie: Consultancy, Honoraria; Novartis: Honoraria; Taiho Oncology: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Boston Biomedical: Consultancy; Astex Pharmaceuticals: Honoraria, Research Funding; Genentech: Research Funding; Apellis Pharmaceuticals: Research Funding.

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. The majority of patients can be cured under the treatment of R-CHOP, but one third of patients will relapse and have a worse prognosis. To exploring a biomarker which can predict clinical behavior may aid in the implementation of novel therapeutic strategies. Inhibitor of DNA-binding 3 (ID3) is a helix loop-helix transcription factor, which negatively regulates the cell proliferation. ID3 mutations occur in 34-68% of Bukitt's lymphoma (BL) and are assiociated with lymphomagenesis. A variety of nonsense and frameshift mutations in ID3 as well as missense mutations in the region encoding the HLH domain reduces or eliminates ID3 protein function in BL. ID3 mutation is rare in DLBCL. The prognostic value of ID3 protein level remains unclear in DLBCL. Therefore, we conducted a retrospective study to investigate the impact of ID3 expression in DLBCL patients. Methods: One hundred DLBCL patients treated at Fudan University Shanghai Cancer Center with available diagnostic biopsy material were identified. ID3 expression was determined and quantified by immunohistochemistry (IHC) with staining intensity and percentage of positive cells (IRS). The IRS cut off value for ID3 positive is equal or more than 6. Kaplan-Meier and life table method were used to calculate progression free survival (PFS) and overall survival (OS). Curves were compared with the long-rank test in both groups. Correlation between the ID3 expression and clinical variables were tested by Pearson Chi Square test and a two-sided P value of Figure Disclosures No relevant conflicts of interest to declare.

Background: The prognosis of relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL) that had progressed after or are ineligible for high dose chemotherapy and autologous stem cell support (HDC-ASCS) is extremely poor (median overall survival of 6.3 months). Cellular based immunotherapy with Chimeric Antigen Receptor T-cell against CD19 (CART-19) has proven high effective in r/r DLBCL with an overall response rate of 52-83% and an overall survival rate of 40%. While r/r DLBCL benefits from CART-19 therapy, a significant number of progress after initial response stresses the need to improve the efficacy of this novel approach. A significant amount of work is being done in improving the manufacturing or activation of CART cells. However, there is few data on lymphoma associated factors that could influence response to CART-19 therapy. Previously we reported an imbalance and dysfunction of pro- and anti-apoptotic proteins, including Bak/Bax, Mcl-1/BCLxL/Survivin and upregulation of inhibitor of apoptosis (IAP) family proteins in rituximab+chemotherapy (R-chemo) resistant DLBCL. Targeting IAPs by small inhibitor LCL161 in lymphoma cells significantly overcame chemotherapy resistance in vitro and in vivo. LCL161 potentiated CART-19 cell cytotoxicity mediated in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma pre-clinical models. Here, we investigate the mechanism of action to enhance immune activation by targeting IAPs in r/r DLBCL. Methods: Primary T-cells were isolated and activated from peripheral blood apheresis samples from consenting adults (healthy human donors or patients with DLBCL) using Pan T-Cell Isolation Kit (MACS) and activated by T-cell Dynabeads human activator CD3/CD28 kit. Cells were expanded in RPMI media supplemented with IL2 for 2 weeks. At day 14, cells phenotype analysis was performed (CD3, CD4, CD8) by flow cytometry. T-cell number was calculated by trypan blue exclusion assay and proliferation was measured by presto blue assay after exposure to LCL161 or control for 48hrs. After exposure, CD4 and CD8 ratios were determined by cellular surface staining. T-cell function assay was determined by intracellular staining with perforin and granzyme B. Regulatory T-cells were determined as CD25+ and FOXP3+ staining cells. T-cell cytolytic activity was accessed by 51Cr release assay using 51Cr labeled rituximab-chemotherapy sensitive (Raji and RL cells) or resistant (Raji 4RH and RL 4RH) cell lines as target cells (effector/target ratio 10/1). To confirm the role of IAP proteins in the effects of LCL161 in T-cell activation, we conducted cytotoxicity assays using XIAP knock out lymphoma cell lines as target cells. Stable XIAP knock out cell lines were generated by CRISPR-Cas9 gene editing system. Lentiviral Cas9 nuclease particles were transduced into Raji and Raji 4RH cells followed by lentiviral XIAP sgRNA transduction. Lentiviral sgRNA non-targeting control was also used as negative control. Result: In vitro exposure of T-cells to LCL161 did not affect cell viability. On the other hand, it led to an increase in CD8+ T-cells and in the CD8:CD4 ratio. In addition, the percentage of regulatory T cells (CD25+ and FOXP3+) was reduced following LCL161 exposure. Using the T-cell function assay, we found that perforin and granzyme B inside T-cell was significantly increased post LCL161 exposure. The presence of LCL161 significantly improved the T-cell killing activity towards rituximab-chemotherapy sensitive and more importantly resistant cell lines. XIAP knock out in target cells resulted in abrogation of the enhance cytotoxicity exhibited by T-cells exposed to LCL161. Conclusion: Our data suggest that besides its previously documented direct anti-tumor activity, LCL161 has dual functions modulating immune cells function. LCL161 improve T-cell function by increasing the number of CD8+ T-cells and intracellular levels of granzyme B and perforin leading to an increase in T-cell cytotoxicity. In addition, LCL161 decreases the number of T-reg cells further enhancing immune activation. Taking together, targeting IAPs would be an attractive approach that potentiate current CART-19 immunotherapy in the clinical setting. Disclosures Hernandez-Ilizaliturri: Astra Zeneca: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Seattle Genetics: Consultancy; Epyzome: Consultancy; Pharmacyclics: Consultancy; Amgen: Consultancy; Gilead: Consultancy.

// Juan J. Gu 1, 2 , Gregory P. Kaufman 3 , Cory Mavis 1, 2 , Myron S Czuczman 4 , Francisco J. Hernandez-Ilizaliturri 1, 2 1 Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA 2 Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA 3 Department of Internal Medicine, Mayo Clinic, Rochester, MN, Celgene Corporation, Summit, NJ, USA Correspondence to: Francisco J. Hernandez-Ilizaliturri, email: francisco.hernandez@roswellpark.org Keywords: bortezomib, B-cell lymphoma, rituximab Received: March 29, 2016 Accepted: December 05, 2016 Published: December 31, 2016 ABSTRACT The ubiqutin-proteasome system (UPS) plays a role in rituximab-chemotherapy resistance and bortezomib (BTZ) possesses caspase-dependent (i.e. Bak stabilization) and a less characterized caspase–independent mechanism-of-action(s). Here, we define BTZ-induced caspase-independent cell death pathways. A panel of rituximab-sensitive (RSCL), rituximab-resistant cell lines (RRCL) and primary tumor cells derived from lymphoma patients ( N = 13) were exposed to BTZ. Changes in cell viability, cell-cycle, senescence, and mitotic index were quantified. In resting conditions, RRCL exhibits a low-proliferation rate, accumulation of cells in S-phase and senescence. Exposure of RRCL to BTZ reduces cell senescence, induced G2-M phase cell-cycle arrest, and is associated with mitotic catastrophe. BTZ stabilized p21, CDC2, and cyclin B in RRCL and in primary tumor cells. Transient p21 knockdown alleviates BTZ-induced senescence inhibition, G2-M cell cycle blockade, and mitotic catastrophe. Our data suggest that BTZ can induce apoptosis or mitotic catastrophe and that p21 has a pivotal role in BTZ activity against RRCL.

Anthracycline-induced cardiotoxicity influences treatment selection and may negatively affect clinical outcomes in lymphoma patients. While epirubicin induced cardiotoxicity less often than the same dose of doxorubicin in breast cancer, higher doses of epirubicin are required in lymphoma regimens for equivalent efficacy. Whether a higher dosage of epirubicin also induces cardiotoxicity less often than doxorubicin in lymphoma remains unknown. We therefore administered 6-8 cycles of cyclophosphamide, vincristine and prednisone (CEpOP) +/- rituximab (R) with either epirubicin (CEpOP) or doxorubicin (CHOP) to patients (N=398) with untreated diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma grade 3 (FLG3). Left ventricular ejection fraction (LVEF) and high-sensitivity serum cardiac troponin T (HsTnT) were assessed at baseline and after 4 cycles of treatment. Epirubicin (70 mg/m2/dose) was equivalent to doxorubicin (50 mg/m2/dose) in terms of 3-year progression-free survival. The risk of decreased LVEF was similar between the two regimens. CEpOP+/-R induced HsTnT elevation less often than CHOP+/-R. We conclude that CEpOP+/-R is a more acceptable regimen with short-term efficacy similar to CHOP+/-R in lymphoma patients. Longer follow-up is needed to monitor the risk of cardiac dysfunction and determine whether differences in the induction of elevated HsTnT between epirubicin and doxorubicin justify changes in clinical practice.

Background: The development of drugs targeting BTK and BCL2 has dramatically improved the therapeutic landscape in chronic lymphocytic leukemia (CLL). However, resistance to these agents has been reported due to non-recurrent changes in oncogenic pathways and gene expression signatures. The bromodomain and extra terminal (BET) family proteins (BRD2, BRD3, BRD4 and BRDT) are epigenetic reader proteins that recognize acetylated lysine residues in histones. They play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Of these, the BRD4 protein is highly enriched in super enhancers and regulates transcription of relevant oncogenes such as MYC, CDK genes, cyclin-D1, BCL2 and MCL-1. BRD4 inhibitors (BRD4i) block the transcription of these key oncogenes through the displacement of BRDs and other epigenetic modifiers from chromatin. In this study, we investigated the preclinical activity of two BRD4i, AZD5153 and PLX51107, and their cooperative role with the BCL2 inhibitor venetoclax and BTK inhibitor ibrutinib in CLL. Methods: Primary tumor cells were isolated from 36 CLL patients. Peripheral blood mononuclear cells were isolated via histopaque-1077 (Sigma-Aldrich, St Louis MO) from peripheral blood apheresis samples from consenting adults under RPCCC protocol CIC-01-16. B-cells were then isolated from enriched lymphocytes by MACS separation using a human B-cell Isolation Kit II. CLL cells were seeded at 3 x106/mL in a 384 well tissue culture plates and exposed to different doses of AZD5153 (1-20uM) or PLX51107 (1-20uM) as a single agent or in combination with venetoclax (1-100nM) or ibrutinib (50-1000nM) for 48 and 72hrs (Selleck Chemicals, Houston TX). Cell viability was assessed using Cell Titer-glo (Promega, Madison WI) with all data being normalized to untreated controls. Synergy was calculated using Calcusyn (Cambridge, UK) software to determine Coefficient of Synergy (CI) values. In addition, CLL cells were plated in 6 well plates and exposed to AZD5153 or PLX51107 alone or in combination with either venetoclax or ibrutinib for 24 hrs. Induction of apoptosis and changes in B-cell receptor signaling, BCL2 and NF-kB pathway related proteins were evaluated by flow cytometry or Western blotting respectively. Results: The study included 33 treatment naïve and 3 relapsed/refractory CLL patients. Deletion 11q was detected in 6 patients and del 17p was seen in 1 patient. Both AZD5153 and PLX51107 displayed time and dose dependent induction of apoptosis of CLL cells. Activity was observed in both treatment naïve and relapsed/refractory samples. Moreover, activity was observed in del 17p or ATM deletion samples. Synergistic activity was observed between BRD4is and venetoclax. PLX51107 exhibited synergy with venetoclax in 83% of the samples tested. Similarly, AZD5153 exhibited synergistic effects when combined with venetoclax in 61% of the samples. To a lesser degree, BRD4is exhibited synergistic activity when combined with ibrutinib, (44% with PLX51107 and 39% with AZD5153). Induction of apoptosis was observed in CLL cells exposed to either BRD4i. Morevoer, an increase in the percentage of apoptosis was observed when BRD4i was combined with venetoclax. Western blotting showed a accumulation of p21, reduced levels of C-MYC, as well as modulation of BCL-2 family member proteins such as BCL2, MCL-1 following BRD4i ex vivo exposure. Conclusions: Our data indicate that BRD4 inhibition results in apoptosis of primary tumor cells isolated from patients with various subtypes of CLL. PLX51107 and AZD5153 exhibit synergistic effects when combined with venetoclax and to a lesser degree with ibrutinib. Modulation of BCL-2 family members following BRD4 inibition could play a role in the synergistic activity observed in our studies. A better understanding of the key signaling pathways involved in proliferation and survival of CLL cells impacted by BRD4i in combination with venetoclax and ibrutinib would provide a proof-of-concept rationale for studies validating BRD4i as epigenetic approach to target BCR signaling in CLL. Disclosures Hernandez-Ilizaliturri: Karyopharm: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Gilead: Consultancy; Pharmacyclics: Consultancy; Seattle Genetics: Consultancy; Astra Zeneca: Consultancy; Epyzome: Consultancy.

Purpose: In patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), resistance to rituximab confers chemotherapy-resistance and poor response to salvage regimens. MDM2 is an E3 ligase which regulates the degradation of multiple cellular protein targets, but is known best as the major negative regulator of the tumor suppressor, p53. Recently, we demonstrated that MX69, a MDM2 inhibitor, re-sensitized resistant DLBCL cell lines to chemotherapy agents or small inhibitors (i.e. Venetoclax) in vitro by blocking the MDM2 protein-XIAP RNA interaction, led to both MDM2 and XIAP degradation, thus establishing the feasibility of this approach for overcoming rituximab and chemotherapy resistance in DLBCL. We now report the activity of Idasanutlin, an investigational Nutlin-family MDM2 antagonist, in pre-clinical models of R-chemo resistant B-cell lymphoma. Methods: We used a panel of rituximab sensitive and R-chemo resistant B-cell lymphoma cell lines representing activated B-cell-like (ABC) DLBCL (TMD8, U2932); germinal center B-cell like (GCB) DLBCL (RL, RL 4RH, OCILy2); double hit DLBCL (DHL4, DHL6, DOHH2, VAL, ROS50), and Burkitt's lymphoma (Raji and Raji 4RH). Cells were exposed to escalating doses of Idasanutlin as single agent and in combination with various chemotherapeutic agents and small molecule inhibitors for 24, 48 and 72 hrs. Differences in cell viability were evaluated by PrestoBlue. IC50 was calculated by GraphPad and Coefficient of synergy was calculated using CalcuSyn. Low mitochondrial potential was detected by staining cell with DiOC6 followed by flow cytometric analysis. Western blot was used to detect changes in MDM2, XIAP and PUMA expression. Results: In vitro exposure to Idasanutlin demonstrated dose- and time-dependent cell death in DLBCL cell lines, IC50 ranged from 0.7uM to 63.07uM at 48h. Idasanutlin at the dose of 1uM was able to disrupt mitochondria and lowered the mitochondrial membrane potential in both ABC and GCB cell lines at 48 h. Idasanutlin reduced the expression of MDM2 in TMD8, U2932, VAL, OCILy2 DHL4 and ROS50 cell lines. XIAP expression was reduced in VAL and DHL4. PUMA, the downstream product of p53 activation, was increased after idasanutlin exposure in all cell lines. Idasanutlin demonstrated significant synergy with chemotherapeutic reagents (etoposide and vincristine), proteasome inhibitors (carfilzomib and ixazomib), Bcl-2 inhibitor venetoclax and Bruton tyrosine kinase inhibitor ibrutinib. Conclusion: Idasanutlin showed promising cell-of-origin agnostic anti-tumor activity as a single agent and in combination with several small molecule inhibitors in pre-clinical models of DLBCL. It decreased MDM2 and XIAP protein level and induced PUMA expression. Its unique mechanism of action through p53 modulation makes it an attractive partner for use in a variety of clinical settings in B-cell lymphomas. Citation Format: Juan J. Gu, Samuel Thompson, Cory Mavis, Pallawi Torka, Francisco Hernandez-Ilizaliturri. MDM2 inhibitor Idasanutlin potentiate therapeutic agents in Diffuse Large B-Cell Lymphoma by targeting MDM2 and XIAP [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 2404.

BackgroundCancer has become increasingly prevalent in China over the past few decades. Among the factors that determine the quality of life of cancer patients, pain has commonly been recognized as a most critical one; it could also lead to the ineffective treatment of the cancer. Driven by the need for better pain management for cancer patients, our research team developed a mobile-based Intelligent Pain Management System (IPMS). ObjectiveOur objective was to design, develop, and test the IPMS to facilitate real-time pain recording and timely intervention among cancer patients with pain. The system’s usability, feasibility, compliance, and satisfaction were also assessed. MethodsA sample of 46 patients with cancer pain symptoms were recruited at the Oncology Center of Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Chongming Branch (hereinafter referred to as “the Oncology Center”). In a pretest, participants completed a pain management knowledge questionnaire and were evaluated using the baseline cancer pain assessment and Karnofsky Performance Status (KPS) evaluation. The participants were then randomly assigned into two groups (the trial group and the control group). After a 14-day trial period, another round of cancer pain assessment, KPS evaluation and pain management knowledge assessment were repeated. In the trial group, the data were fully automatically collected by the IPMS. In the control group, the data were collected using conventional methods, such as phone interviews or door-to-door visits by physicians. The participants were also asked to complete a satisfaction questionnaire on the use of the IPMS. ResultsAll participants successfully completed the trial. First, the feasibility of IPMS by observing the number of daily pain assessments recorded among patients was assessed. Second, the users’ satisfaction, effectiveness of pain management, and changes in the quality of their lives were evaluated. All the participants gave high satisfaction score after they used IMPS. Both groups reported similar pain scores and KPS scores at the baseline. At the end of the trial, the mean pain score of the trial group was significantly lower than of the control group (P

Background: Although first-line chemo-immunotherapy cures a significant number of diffuse large B-cell lymphoma (DLBCL) patients, long term survival is only observed in 30% or 41% of patients treated with second-line therapy followed with high dose chemotherapy and autologous stem cell support (HDC-ASCS) or chimeric antigen receptor anti-CD19 T-cell therapy (CART-19) respectively. The use of third-line therapy with chemotherapy drugs or current available small molecule inhibitors for relapsed/refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) is associated with a poor response rate and a median survival of 6.3 months, stressing the need to identify druggable biomarkers associated with therapy resistance and poor clinical outcomes. Methods: To this end, we identified patients with rel/ref DLBCL that failed at least 2 prior lines of therapy treated at our Institute. Demographic, clinical and pathological characteristics were identified. Archived pathological material from the biopsy performed at the second relapse/progression was obtained for each patient. Using the Nanostring nCounter Analysis System (Nanostring Technologies, Seattle, WA), gene expression profiling (GEP) was performed for each sample as previously described using a custom designed code set containing 770 genes. Analysis and normalization of the raw Nanostring data was performed using nSolver Analysis Software v1.1. Raw counts were normalized to internal levels of 7 reference genes: CNOT2, GAPDH, HPRT1, PHGDH, SUMO2, SYS1 and WDR45L. A background count level was estimated using the average count of the 8 negative control probes in every reaction plus 2 standard deviations. Correlation between clinical outcomes (response rate to third line therapy, progression free-survival [PFS] and overall survival [OS]) and IPI score, clinical characteristics and GEP. Subsequently and based on the GEP results, we evaluated the expression of Bcl-XL and other Bcl-2 family proteins in a panel of DLBCL cell lines. Immuno-precipitation (IP) studies were conducted to determine protein-protein interaction between Bcl-XL and other Bcl-2 related proteins. In addition, cell lines were exposed to A1331852, a novel Bcl-XL inhibitor at different doses alone or in combination with chemotherapy drugs. Cell viability was evaluated using Presto Blue assay and IC50 values were calculated using the GraphPad Prism6 software. Results: A total of 53 rel/ref DLBCL were identified that received third line therapy at our Institute. The median age was 59yrs. The median PFS and OS was 23 months and 21 months respectively. Over-expression of BCL-XL RNA levels was associated with a shorter OS (P=0.015, HR=1.17). Pre-clinically higher levels of Bcl-XL as determined by western blotting were found in lymphoma cell lines resistant to chemotherapy agents in vitro. IP studies demonstrated a strong interaction between Bcl-XL and BIM in chemotherapy resistant cell lines. In vitro exposure of DLBCL cell lines to A1331852 resulted in dose- and time-dependent cell death. Moreover, synergistic activity was observed when A1331852 was combined with chemotherapy drugs (doxorubicin, vincristine and etoposide). Conclusion: Our data suggests that high expression level of BCLxL was associated with inferior prognosis in the refractory and/or relapsed DLBCL patients. Inhibition of Bcl-XL results in cell death of rituximab-chemotherapy resistant lymphoma cell lines and potentiates the anti-tumor activity of chemotherapy agents. Selective targeting of Bcl-XL may be a novel therapeutic strategy for rel/ref aggressive B-cell lymphoma. Disclosures No relevant conflicts of interest to declare.

Background: Although >90% of Burkitt Lymphoma (BL) patients will be cured by intensive multi-agent immunochemotherapy, relapsed and refractory BL patients have poor prognoses demonstrating the need to identify novel therapies. Myeloid cell leukemia-1 (MCL-1) is a pro-survival protein within the BCL-2 family that has been investigated as a therapeutic target in several leukemia and lymphoma pre-clinical models and clinical studies. Analysis of pediatric BL tumors has implicated MCL-1 in BL pathogenesis (Giulino-Roth et al., Blood 2012) and targeting MCL-1 may be effective in MYC-driven lymphomas (Kelly et al., Genes Dev 2014). High MCL-1 expression has also been associated with therapy resistance and can confer resistance to BH3 mimetic agents (Carrington et al., Cell Death Differ 2017). AMG176, a selective MCL-1 inhibitor, is an effective promoter of apoptosis and is currently under investigation in clinical trials for relapsed and refractory multiple myeloma, lymphoma, and acute myeloid leukemia. Hypothesis: We hypothesized that MCL-1 inhibition with AMG176 will promote apoptosis and increase sensitivity of BL therapy-sensitive (TSCL) and therapy-resistant (TRCL) cell lines to other chemotherapy drugs. Methods: A panel of TSCLs (Raji, Ramos and Daudi) and one TRCL (Raji 4RH) BL cell lines were exposed to increasing concentrations of AMG176 as a single agent and in combination with Doxorubicin or Venetoclax at different time points. Cell viability was analyzed using PrestoBlue. Synergy in drug combination exposures was determined by Calcusyn software and reported as a combination index (CI) value with values increasingly Results: Single agent AMG176 exposure of BL cell lines exhibited a dose and time-dependent decrease in viable cells. 48hr IC50 values of Ramos (0.3097uM) and Raji (1.652uM) showed an increased sensitivity to AMG176 as compared to Daudi (9.616uM) and Raji 4RH (12.45uM). Exposure of cells to AMG176 also resulted in altered expression levels of pro-survival and pro-apoptotic proteins of the BCL-2 family proteins. Co-immunoprecipitation performed after 48hrs of AMG176 exposure exhibited a decrease in MCL-1:BIM and MCL-1:BAK complexes in Raji and Ramos cells promoting a pro-apoptotic environment. After 48hr exposure to AMG176, induction of apoptosis was observed in a dose-dependent manner in all cell lines as evidenced by increased PARP cleavage on western blot and AV-Sytox positivity on flow cytometry. To investigate the ability of MCL-1 inhibition to enhance the activity of cytotoxic chemotherapy, BL cells were exposed to AMG176 in combination with Doxorubicin. Daudi and Raji exhibited strong synergy with peak synergistic activity observed at 72hrs (Daudi CI values Conclusion: MCL-1 inhibition with AMG176 demonstrates dose- and time-dependent anti-lymphoma activity impairing in vitro proliferation and inducing apoptosis in BL cells. AMG176 exhibits synergistic activity in combination with cytotoxic chemotherapy in both therapy-sensitive and therapy-resistant BL cell lines and synergistically enhances response to the BH3 mimetic agent Venetoclax. These findings highlight the potential of MCL-1 inhibition as a therapeutic option in BL. This work was supported by T35 NIH training grant (T35AI089693). Disclosures No relevant conflicts of interest to declare.

Purpose: The combination of rituximab and chemotherapy has improved overall survival of diffuse large B-cell lymphoma (DLBCL) patients in recent decades. Relapsed/refractory DLBCL patients previously treated with rituximab-containing regimen had significantly poorer response to salvage therapy. To study the mechanisms of this resistance, our laboratory developed several rituximab resistant cell lines. Previous study from our group found demonstrated an aberrant imbalance in the levels of pro- and anti-apoptotic proteins in these cell lines, including Bak/Bak, Mcl-1/BCLxL/Survivin and inhibitor of apoptosis (IAP) family proteins resulting in acquired chemotherapy resistance. MDM2 is an E3 ligase which regulates the degradation of multiple cellular protein targets such as pRB, HIF-1, p73, NF-kB, and E2F1 as well as FOXO3a. It is also the major negative regulator of p53. Recently, we found that MX69 (a MDM2 inhibitor), which blocks the MDM2 protein-XIAP RNA interaction, led to both MDM2 and XIAP degradation, and induced apoptosis-dependent cell killing activity. Moreover, MX69 re-sensitized resistant DLBCL cell lines to chemotherapy agents or small inhibitors (i.e. Venetoclax) in vitro. However, MX69 cannot be used clinically stressing the need to use a clinically available MDM2 inhibitor. To this end, we evaluated Idasanutlin, an investigational Nutlin family MDM2 antagonist, in B-cell lymphoma pre-clinical models. Methods: We used a panel of different DLBCL subtypes cell lines including activated B-cell lymphoma cell lines (TMD8, U2932); germinal center B-cell lymphoma (DOHH2, VAL, OCILY2, DH4, DH6, ROS50); rituximab-sensitive cell lines (Raji and RL); and rituximab/-resistant cell lines (Raji 4RH and RL 4RH). Cells were exposed to escalating doses of Idasanutlin (1nM-100µM) without or with other chemotherapeutic agents for 24, 48 and 72 hrs. Differences in cell viability were evaluated utilizing PrestoBlue. IC50 was calculated by GraphPad and Coefficient of synergy was calculated using CalcuSyn. Low mitochondrial potential was detected by staining cell with DiOC6 10ng/ml for 30 minutes and followed by flow cytometric analysis. Western blot was used to detect the changes of MDM2, XIAP and PUMA expression before and after exposure to Idasanutlin 1uM for 24h. Results:In vitro exposure Idasanutlin to DLBCL cell lines demonstrated a dose- and time-dependent cell death. IC50 dosage of the cells was ranged from 0.7uM to 63.07uM at 48h. Low dose of Idasanutlin (1uM) was able to disrupt mitochondria and induced low mitochondrial membrane potential in both ABC and GCB cell lines. At molecular level, Idasanutlin reduced expression level of MDM2 in TMD8, U2932, VAL, OCILy2 DHL4 and ROS50 cell lines. XIAP was reduced in Val and DHL4. Meanwhile, PUMA, the downstream of p53 activation, was increased after Idasanutlin exposure. Idasanutlin enhanced the anti-tumor activity of proteasome inhibitors (carfilzomib, ixazomib), Bcl-2 inhibitor venetoclax and Bryton kinase inhibitor ibrutinib. Conclusion: Idasanutlin showed potent anti-tumor activity as a single agent in DLBCL pre-clinical setting. Idasanutlin was able to decrease cellular mitochondrial membrane potential. At molecular level, Idasanutlin decreased MDM2 and XIAP protein level and induced PUMA expression. Moreover, Idasanutlin enhanced anti-tumor activities of other small inhibitors. Taking together, Idasanutlin could be used as a novel and promising drug in the clinical setting of DLBCL with relapsed/refractory disease. Disclosures No relevant conflicts of interest to declare.

Purpose: The results of the prospective multicenter phase III Collaborative trial in relapsed aggressive lymphoma (CORAL) study stressed the need to identify cellular pathways associated with poor clinical outcomes and to develop novel therapeutic strategies for DLBCL. We previously demonstrated that over-expression of HKII contribute to the acquirement of resistance to rituximab and chemotherapy agents in DLBCL B-cell lymphoma in pre-clinical models. Using gene expression profiling, we also demonstrated that higher mRNA levels of HKII correlated with a shorter progression free survival (PFS) and overall survival (OS) in previously untreated DLBCL in the pre- and post-rituximab era. In current study, we aimed to assess HKII expression by immunohistochemistry (IHC) and analyze the prognostic significance in 97 diffuse large B cell lymphoma patients pre- and post-rituximab era treated in a different Country/Institution. Methods: We retrospectively identified 97 newly diagnosed DLBCL treated at Fudan University Shanghai Cancer Center, Shanghai, China from March 2009 to December 2012. Clinical, demographic and pathological characteristics for each patient were collected. Patients were treated with standard doses of CHOP (N=41) or rituximab and CHOP (N=56). Pathological archived material was submitted for immunohistochemistry (IHC) for the determination of cell of origin (COO) (Han's algorithm) and HKII levels. Co-relation between HKII expression and COO or impact on overall response rate (ORR), PFS and OS were analyzed. Result: Demographic and clinical characteristic of gender, stage and IPI score distribution were similar between patients treated with CHOP or R+CHOP. As expected, the clinical outcome of DLBCL patients treated with R+CHOP was superior than those treated with CHOP. HKII expression was observed in 62 DLBCL patients (63.9%). There was a correlation between HKII expression and COO. Expression of HKII was observer in 84.6% of non-germinal center B-cell (non-GCB) DLBCL and 60% of GCB DLBCL (P=0.027). There was no correlation between HKII expression and ORR or PFS in DLBCL patients treated with CHOP or R-CHOP. In patients treated with CHOP chemotherapy, HKII expression was associated with an inferior OS, 100% (HKII negative) vs. 67% (HKII positive) (P=0.025). In rituximab era, additionally rituximab to CHOP results significantly improved OS in HKII positive patient compared to CHOP only patients (91% vs 67%, P=0.05), but R+CHOP did not change OS between HKII negative patients. Conclusion: As previously observed, HKII expression is more prevalent in non-GCB DLBCL patients. In pre-rituximab era, HKII expression was associated with a poor clinical outcome after CHOP chemotherapy. The negative effect of HKII expression appears to be overcome by the addition of rituximab to CHOP. (This work was supported by National Natural Science Foundation of China (81670177 and 81773203)). Disclosures No relevant conflicts of interest to declare.

Purpose: Despite T cell lymphoma (TCL) responses well at the initial chemotherapy, the outcome of TCL remains poor when it becomes systematic disease with refractory or relapse (r/r) after several cycle of treatment. Constitutively activated PI3K-mTOR pathway was found variety of cancers including TCL, and dual inhibition of PI3k-mTOR has been shown preclinically and clinically successful in B-cell lymphoma. However, the activity of dual inhibition of PI3k-mTOR in T-cell malignancies remain utterly unknown. GSK458 is a potent oral dual inhibitor of pan PI3K (α, β, γ and δ) and mTOR (mTOR1 and mTOR2). Preclinical study showed GSK458 had broad antitumor activity in vitro and in vivo. In 2016, phase I clinical trial of GSK458 was competed and the dosage was durable in multiple tumor types. Here we extensively characterized the activity and the mechanism of action of GSK458 in preclinical model of T-cell lymphoma alone and in combination therapies. Methods: This study included a panel of T cell lymphoma lines as well as primary samples derived from lymphoma patient, including J45 and SupT-1 are PTCL as T-cell lymphoblastic lymphoma; whereas MJ, HH and H9 are CTCL represented Mycosis fungoides (MF), aggressive leukemic MF and the most aggressive form of CTCL Sezary syndrome (SS) respectively. In vitro viability assay was performed as single agent of GSK458 and in combination with other drugs. Differences in cellular metabolic difference were examined by ATP levels, low mitochondria potential and glucose update utilizing Cell-Titer Glo assays, DiOC6 and 2-NDG flow analysis, respectively. Cellular apoptosis and cell cycle distribution were evaluated by Annexin V and propidium iodide staining followed by flow cytometry. PI3K and mTOR downstream pathway phosphorylation status were detected by flow cytometry. Apoptosis proteins were detected by western blot. The regulative T cells were evaluated by flow cytometry. Results: GSK458 had potent anti-tumor activity as single agent and potentiated the activity of chemotherapy or small inhibitors, such as doxorubicin, carboplatin, venetoclax, brentuximab, romdespsin and proteasome inhibitors in TCL pre-clinical models. GSK458 sufficiently blocked the downstream targets of PI3K-mTOR, such as phosphorylation status of Akt serine473 and threonine308; phosphorylation of mTOR and GSK3β. At metabolic level, GSK458 reduced ATP production, decrease glucose uptake, lowered mitochondrial membrane potential and triggered apoptosis in TCL. Moreover, GSK458 arrested the cell cycle at G1. In our study, we found after exposure to GSK458, the percentage of regulatory T cell among the total CD4+ T cells were decreased. Conclusion: Based on these preclinical results, GSK458 appeared as a novel and promising drug that is possible to develop clinical trial in T-cell lymphoma. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) Citation Format: Juan J. Gu, Lianjuan yang, Jing He, Weina Shen, Cory Mavis, Francisco Hernandez-Ilizaliturri. GSK458, a novel oral dual PI3K/mTOR Inhibitor, is effective in preclinical model of T-cell lymphoma alone and in combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3919.

To further develop therapeutic strategies targeting the proteasome system, we studied the antitumor activity and mechanisms of action of MLN2238, a reversible proteasome inhibitor, in preclinical lymphoma models. Experiments were conducted in rituximab-chemotherapy-sensitive cell lines, rituximab-chemotherapy-resistant cell lines (RRCL), and primary B-cell lymphoma cells. Cells were exposed to MLN2238 or caspase-dependent inhibitors, and differences in cell viability, alterations in apoptotic protein levels, effects on cell cycle, and the possibility of synergy when combined with chemotherapeutic agents were evaluated. MLN2238 showed more potent dose-dependent and time-dependent cytotoxicity and inhibition of cell proliferation in lymphoma cells than bortezomib. Our data suggest that MLN2238 can induce caspase-independent cell death in RRCL. MLN2238 (and to a much lesser degree bortezomib) reduced RRCL S phase and induced cell cycle arrest in the G2/M phase. Exposure of rituximab-chemotherapy-sensitive cell lines and RRCL to MLN2238 potentiated the cytotoxic effects of gemcitabine, doxorubicin, and paclitaxel and overcame resistance to chemotherapy in RRCL. MLN2238 is a potent proteasome inhibitor active in rituximab-chemotherapy-sensitive and rituximab-chemotherapy-resistant cell models and potentiates the antitumor activity of chemotherapy agents and has the potential of becoming an effective therapeutic agent in the treatment of therapy-resistant B-cell lymphoma.

Background: Despite significant gains achieved in the treatment of Burkitt lymphoma (BL), current multi-agent immunochemotherapeutic regimens lead to high rates of acute toxicity, and relapsed/refractory disease still represents a significant hurdle with survival expected in only about 20-30% of such patients. Novel targeted therapeutic approaches are necessary to reduce treatment related toxicity in the up-front setting and improve survival in the relapsed/refractory setting. Analyses of genomic abnormalities in BL have identified increased activation of the PI3K/Akt/mTOR pathway in BL, induced by tonic B-cell receptor signaling and increased expression of Myc induced microRNAs (miRs), as having a significant role in Burkitt lymphomagensis. Additionally, recent reports have implicated higher expression of PI3K activating, Myc induced miRs in pediatric patients with a higher risk of relapse. While focused targeting of PI3K with the PI3K-delta isoform specific inhibitor idelalisib has led to significant activity in indolent B-cell lymphomas, limited activity has been noted in the setting of more aggressive forms. A broader inhibition of both upstream and downstream components of the pathway may exhibit more significant anti-lymphoma activity. To this end, we investigated the in vitro effects of PI3K/Akt/mTOR pathway inhibition with the dual pan-PI3K/mTOR inhibitor Omipalisib (GSK458) in chemotherapy-sensitive and -resistant BL cell line models. Methods: The in vitro effect of omipalisib was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell viability following exposure to omipalisib alone and in combination with cytotoxic chemotherapeutic agents was analyzed using Cell-Titer Glo and Alamar Blue assays. Apoptosis was analyzed using western blotting for PARP and by flow cytometry with Annexin V-propidium iodide staining. Downstream targets in the PI3K/Akt/mTOR pathway were analyzed using western blotting. Cell cycle analysis was performed by flow cytometry using propidium iodide staining. Synergistic activity of combination exposures was determined by calculation of a combination index using CalcuSyn software. Results: In vitro exposure of BL cell lines to omipalisib in concentrations ranging from 0.05μM to 50μM for 24, 48 or 72 hours resulted in a dose and time dependent decrease in viable cells with significant activity noted at even low nM concentrations (48 hour IC50 values: Raji=1.2μM, Raji 4RH=0.02μM, Raji 8RH=1.9μM, Ramos=0.01μM, Daudi=0.01μM). Flow cytometry for Annexin V and propidium iodide, after 72 hours of single agent exposure to omipalisib, showed a marked induction of apoptosis in all cell lines. For example, at an omipalisib concentration of 200nM, the percentage of Annexin V positive cells were Raji=40.7%, Raji 4RH=4.4%, Raji 8RH=41.5%, Ramos=59.4% and Daudi=46.9%. Approximately ten-fold higher omipalisib concentrations were required to induce similar degrees of apoptosis in the chemotherapy resistant Raji 4RH cell line compared to chemotherapy sensitive cell lines. Western blotting for downstream targets of the PI3K/Akt/mTOR pathway, including S6 and GSK3Β, showed a reduction in phosphorylation after 30 minutes of exposure to omipalisib in all cell lines. Determination of cell cycle progression following exposure to omipalisib for 72 hours at concentrations ranging from 0.006μM to 25μM showed dose-dependent cell cycle arrest in G1 phase in all cell lines; however the chemotherapy resistant Raji 4RH cells arrested in G2/M at higher concentrations. When BL cells were exposed to omipalisib in combination with either doxorubicin or dexamethasone, synergistic anti-tumor activity was observed in all cell lines tested. Conclusion: Inhibition of PI3K and mTOR by the dual inhibitor omipalisib suppresses activation of the PI3K/Akt/mTOR pathway leading to impaired BL cell proliferation with G1 cell cycle arrest and induction of apoptosis in chemotherapy-sensitive and -resistant cell line models of BL. Inhibition of the PI3K/Akt/mTOR pathway with omipalisib also increases the in vitro response to cytotoxic chemotherapeutic agents. Our findings note the pre-clinical activity of PI3K/Akt/mTOR pathway inhibition in BL and highlight the relevance of pursuing PI3K/Akt/mTOR pathway inhibition as a potential therapeutic option in BL. Disclosures No relevant conflicts of interest to declare.

Preclinical models of chemotherapy resistance and clinical observations derived from the prospective multicenter phase III collaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated that primary refractory/relapsed B cell diffuse large B cell lymphoma has a poor clinical outcome with current available second-line treatments. Preclinically, we found that rituximab resistance is associated with a deregulation on the mitochondrial potential rendering lymphoma cells resistant to chemotherapy-induced apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Posttranscriptional histone modification plays an important role in regulating gene transcription and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-proliferation activity in cancer cells. To this end, we studied the anti-proliferation activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, vorinostat) in rituximab–chemotherapy-resistant preclinical models. A panel of rituximab–chemotherapy-sensitive (RSCL) and rituximab–chemotherapy-resistant cell lines (RRCL) and primary tumor cells isolated from relapsed/refractory B cell lymphoma patients were exposed to escalating doses of vorinostat. Changes in mitochondrial potential, ATP synthesis, and cell cycle distribution were determined by Alamar blue reduction, Titer-Glo luminescent assays, and flow cytometric, respectively. Protein lysates were isolated from vorinostat-exposed cells, and changes in members of Bcl-2 family, cell cycle regulatory proteins, and the acetylation status of histone H3 were evaluated by Western blotting. Finally, cell lines were pre-exposed to vorinostat for 48 h and subsequently exposed to several chemotherapy agents (cisplatin, etoposide, or gemcitabine); changes in cell viability were determined by CellTiter-Glo® luminescence assay (Promega, Fitchburg, WI), and synergistic activity was evaluated using the CalcuSyn software. Vorinostat induced dose-dependent cell death in RRCL and in primary tumor cells. In addition, in vitro exposure of RRCL to vorinostat resulted in an increase in p21 and acetylation of histone H3 leading to G1 cell cycle arrest. Vorinostat exposure resulted in apoptosis in RSCL cell lines but not in RRCL. This finding suggests that in RRCL, vorinostat induces cell death by alternative pathways (i.e., irreversible cell cycle arrest). Of interest, vorinostat was found to reverse acquired chemotherapy resistance in RRCL. Our data suggest that vorinostat is active in RRCL with a known defective apoptotic machinery, it can active alternative cell death pathways. Given the multiple pathways affected by HDAC inhibition, vorinostat can potentially be used to overcome acquired resistant to chemotherapy in aggressive B cell lymphoma.

Histone deacetylases (HDACs) inhibitors are active in T-cell lymphoma and are undergoing pre-clinical and clinical testing in other neoplasms. Entinostat is an orally bioavailable class I HDAC inhibitor with a long half-life, which is under evaluation in haematological and solid tumour malignancies. To define the activity and biological effects of entinostat in B-cell lymphoma we studied its anti-tumour activity in several rituximab-sensitive or -resistant pre-clinical models. We demonstrated that entinostat is active in rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL) and primary tumour cells isolated from lymphoma patients (n = 36). Entinostat exposure decreased Bcl-XL (BCL2L1) levels and induced apoptosis in cells. In RSCL and RRCL, entinostat induced p21 (CDKN1A) expression leading to G1 cell cycle arrest and exhibited additive effects when combined with bortezomib or cytarabine. Caspase inhibition diminished entinostat activity in some primary tumour cells suggesting that entinostat has dual mechanisms-of-action. In addition, entinostat increased the expression of CD20 and adhesion molecules. Perhaps related to these effects, we observed a synergistic activity between entinostat and rituximab in a lymphoma-bearing severe combined immunodeficiency (SCID) mouse model. Our data suggests that entinostat is an active HDAC inhibitor that potentiates rituximab activity in vivo and supports its further clinical development in B-cell lymphoma.

Background: Intercellular adhesion molecule-1 (ICAM-1) is a cell-surface receptor involving in cell-to-cell adhesion and as well as co-stimulatory molecule involved in T-cell lymphocyte infiltration and activation. In the pre-rituximab era, high level of ICAM-1 was reported to be a prognostic indicator of better clinical outcomes in diffuse large B-cell lymphoma (DLBCL). The value of ICAM-1 expression in the post-rituximab era remains unclear. To investigate the impact of ICAM-1 expression in DLBCL, we conducted a retrospective study in R+CHOP treated patients. Methods: Patients (N=102) with histologically proven DLBCL treated at Fudan University Shanghai Cancer Center with available diagnostic biopsy material were identified. Forty-three patients were treated with CHOP and 59 patients with R+CHOP. ICAM-1 expression was determined by immunohistochemistry (IHC). ICAM-1 expression was quantified by staining intensity or percentage of positive cells. Tumors were considered positive when at least 75% of tumor cells expressed ICAM-1. Progression free survival (PFS) and overall survival (OS) was plotted by Kaplan-Meier method and curves were compared with the long-rank test in both groups. Correlation between the ICAM-1 expression and clinical variables were tested by Pearson Chi Square test and a two-sided P value of Result: ICAM-1 expressionwas detected in 28 (27.5%) DLBCL patients. The rest of the patients tested negative for ICAM-1 (N=74; 72.5%). Differences in ICAM-1 expression were observed according to cell of origin DLBCL subtype. ICAM-1 expression was higher among germinal center B-cell (GCB) DLBCL (41.4%) vs. non-GCB subtype patients (19.6%, P=0.019). The response rates for R-CHOP and CHOP were, respectively, 89.4% and 77.7% (P=0.409) in ICAM-1 positive patients and 92.5% and 73.5% (p=0.027) in ICAM-1 negative patients. As previously described, ICAM-1 expression was associated with an improved PFS/OS (P=0.03/ P=0.05) in CHOP-treated DLBCL. On the other hand, no differences in PFS/OS were observed between ICAM-1 positive or negative DLBCL patients treated with R+CHOP. The addition of rituximab to CHOP chemotherapy resulted in an improved PFS in ICAM-1 negative DLBCL (P=0.018). On the other hand, the clinical outcome of ICAM-1 positive DLBCL patients treated with CHOP chemotherapy was similar than R+CHOP treated patients. Finally, loss of ICAM-1 expression level was found in our rituximab resistant cell lines, along with reduction of rituximab activity. Conclusion: Before therituximab era, ICAM-1 low expression correlated with worse PFS and OS. In our retrospective clinical analysis, we found that rituximab significantly improved the overall response rate and PFS in ICAM-1 negative subset patients. Our data also indicated that ICAM-1 expression level was higher in GCB than non-GCB subtypes, and this may contribute to better rituximab response in GCB cell types. Gradually exposed lymphoma cell to rituximab lost ICAM-1 expression and may contribute to the resistance to rituximab and chemotherapy agents. Further investigation of ICAM-1 expression needed to be study to enhance the rituximab therapy in lymphoma. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Background: Among B-cell lymphoma, double hit lymphoma (DHL) is defined by a MYC/8q24 combined with another recurrent BCL/18q21 breakpoint. DHL most affects elderly patients (median age ranged from 51 to 65 years) and have a poor prognosis with a median overall survival of 0.2-1.5 year. R+CHOP or high-intensity treatment including high-dose chemotherapy followed stem cell transplant yield to suboptimal outcomes. Therefore, there is a need to discover alternative effective agents. Itraconazole, an oral antifungal drug, was reported had potent anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer by interfering with the apoptotic threshold of cancer cells. Its activity in DHL has yet to be defined. Methods: Double hit lymphoma cell lines: DOHH2, VAL and ROS50 were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism 6 software. Loss of ATP, Caspase 3/7 activation and loss of mitochondrial membrane potential (∆ψm) following itraconazole exposure were assessed by Cell Titer Glo, Caspase3 /7 detection kit and DiOC6 staining, respectively. DHL cells were exposed to itraconazole or vehicle combined with various inhibitors, such as BCL-2 inhibitor ABT199, c-MYC inhibitor JQ1, bruton's kinase inhibitor ibrutinib and different generations of proteasome inhibitors (carfilzomib or MLN4924) for 48h and cell viability was determined by Presto Blue assay. Coefficient of synergy index was calculated using the CalcuSyn software. Result: Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition in DHL. In vitro exposure cells to itraconazole resulted in a loss of ATP, loss of mitochondrial membrane potential and activation of caspase 3/7 activities. Itraconazole caused G1 cell cycle arrest and decrease S phase in DHL. Moreover, itraconazole had a strong synergistic anti-tumor effect combined with various inhibitors, including BCL-2 inhibitor ABT199, c-MYC inhibitor JQ1, bruton's kinase inhibitor ibrutinib and different generations of proteasome inhibitors (bortezomib, carfilzomib, Ixazomib or MLN4924). Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against DHL. Disruption of mitochondrial membrane potential and activation of caspases may contribute its antitumor activity against DHL. It is worth noticing that combined itraconazole with DHL specific target inhibitors ABT199 or JQ1 had a strong synergistic efficacy. Our findings offered the first assessment of the efficacy of itraconazole as a novel anticancer agent in double hit lymphoma. The molecular mechanism of action of itraconazole need to be further investigated in the near further. Disclosures No relevant conflicts of interest to declare.

Background: Results from thecollaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated a poor clinical among patients treated with second-line chemo-immunotherapy in the post-rituximab era, stressing the need to identify and/or optimize novel targeted agents. C-myc expression was found correlate with a poor clinical outcome in patients with newly diagnosed or relapsed/refractory DLBCL. Previously, we found that JQ1 overcomes rituximab-chemotherapy resistance in lymphoma pre-clinical models. JQ1 exhibited synergistic activity when combined with chemotherapy agents. We also demonstrated that hexokinase II was highly expressed in rituximab resistant cell lines (RRCL), and its expression was associated with a deregulation in the glucose metabolism and an increase in the apoptotic threshold leading to chemotherapy resistance. In our current work, we evaluated molecular studies dissecting the cellular pathways affected by JQ1. Methods: A panel of rituximab-sensitive (RSCL) and RRCL cell lines were exposed to escalating doses of JQ1 (0-8μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism 6 software. Loss of mitochondrial membrane potential (∆ψm) following JQ1 exposure was assessed by DiOC6 staining. Changes in c-MYC, HKII, HKI, Voltage dependent anion channel protein (VDAC) and p21 expression levels were determined by western blotting. HKII was silencing using siRNA interference in RRCL (Raji-4RH) and changes in HKII, HKI, VDAC, c-MYC and p21 protein expression was determined. HKII knockdown or scramble Raji-4RH cells were exposed to different chemotherapy agents and JQ1; cell viability and mitochondrial membrane potential was accessed. Result:JQ1 induced a dose-and time- dependent cell death in cell lines. In vitro exposure cells to JQ1 resulted in a loss of mitochondrial potential and induced G1 cell cycle arrest. In vitro exposure to JQ1 decreased c-MYC and HKII expression levels and induced of p21 expression. Complete silencing of HKII re-sensitized cell to chemotherapy (doxorubicin, vincristine and bortezomib). On the other hand, HKII knockout abrogated JQ1 anti-tumor activity. Conclusion: Our data suggests that JQ1 had anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The inhibition of HKII following JQ1 exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic effect. Our finding highlights a better understanding in the molecular events triggered by JQ1 as a potential role in the treatment of B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

Background: Burkitt lymphoma (BL) is the most common NHL type in children. Although treatment for pediatric BL has improved significantly, there is an urgent need for novel therapies that reduce the toxicity of modern treatment regimens and improve on the dismal survival observed in the relapsed/refractory setting where only about 20-30% of patients survive their disease. Recent reports have implicated co-activation of c-Myc and PI3K in Burkitt lymphomagenesis. Genomic analysis of recurrent oncogenic mutations in BL have identified tonic B-cell receptor signaling and the over-expression of Myc induced microRNAs from the MIR17-92 family, e.g. mir17 and mir19, as possible mechanisms of PI3K activation in BL. Mir17 and mir19, have been implicated in Burkitt lymphomagenesis and their overexpression may be associated with a higher risk of relapse. The protein phosphatase and tensin homolog (PTEN) is a major regulator of PI3K pathway activation. MIR17-92 cluster members have been shown to target PTEN leading to increased PI3K activation. We have previously identified increased expression of mir17 and mir19 along with increased activation of AKT in cell line models of chemotherapy resistant BL suggesting a potential mechanism for increased resistance. BET bromodomains interact with chromatin and enhance transcriptional activation of numerous genes including c-Myc. Thus, BET bromodomains represent a promising target in BL. To investigate the effects of inhibition of c-Myc driven activation of the PI3K/AKT/mTOR pathway, we characterized the activity of the highly potent small molecule bromodomain inhibitor JQ1 in chemotherapy sensitive and resistant BL cell lines. Additionally, we analyzed the ability to enhance anti-lymphoma activity of PI3K/Akt/mTOR pathway inhibition in BL by the combination of BET bromodomain inhibition and targeted inhibition of the PI3K/AKT/mTOR pathway. Methods: The in vitro activity of JQ1 was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell Viability following exposure to JQ1 alone and in combination with the PI3K/mTOR inhibitor omipalisib (GSK458)) was analyzed using Cell-Titer Glo or Alamar Blue assays following 24, 48, and 72 hour exposure over a range of inhibitor concentrations. Induction of apoptosis was analyzed using western blotting for cleaved PARP. C-Myc expression following JQ1 exposure was determined by western blot following 48 hour JQ1 exposure. The effect of JQ1 exposure on the expression of c-Myc induced microRNA expression was determined by qRT-PCR in cells exposed to JQ1 for 48hours. Synergy of combination exposures was determined using CalcuSyn to generate combination index (CI) values. Results: In vitro exposure of BL cell lines to JQ1 for 24, 48, and 72 hours resulted in a significant dose and time dependent decrease in viable cells (72 hour IC50 values: Raji 0.12µM, Raji 4RH 1.7µM, Raji 8RH 0.7µM, Ramos 0.22µM and Daudi 4µM). There was an increase in cleaved PARP after 72 hour exposure indicating induction of apoptosis. While single agent effect was seen in the resistant Raji 4RH cell line, activity was noted to primarily occur at the higher end of the dosing. Western blot analysis demonstrated a reduction in c-Myc expression following exposure to JQ1 1µM for 24 hours (relative band intensity normalized to control: Raji=0.12, Raji 4RH=0.18, Raji 8RH=0.11). qPCR showed a reduction in Mir17 relative transcription levels after 48 hours of exposure to JQ1 2.5mM (relative expression compared to control: Raji=0.72, Raji 4RH=0.98, Raji 8RH=0.83, Ramos=0.57, Daudi=0.46). When combined with omipalisib, an increased effect on cell viability was noted. The combination effect was noted to be synergistic (CI Conclusion: In vitro inhibition of BET bromodomains with JQ1 results in a decrease in c-Myc expression and a decrease in c-Myc dependent miR expression with impaired proliferation and induction of apoptosis in chemotherapy-sensitive and -resistant BL cell lines. Augmented, and in some cases synergistic, activity is also noted with dual inhibition of BET bromodomains and the PI3K/Akt/mTOR pathway in BL cell lines. Disclosures No relevant conflicts of interest to declare.

Background: Relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy have poor clinical outcome, according to the results from collaborative trial in relapsed aggressive lymphoma (CORAL) study. It stresses the need to identify and/or optimize novel targeted agents. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). Itraconazole, an oral antifungal agent, was reported had novel anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer. In our current work, we define and characterize the anticancer activity of itraconazole in preclinical rituximab-sensitive or -resistant lymphoma models. Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism6 software. Loss of mitochondrial membrane potential (∆ψm) following itraconazole exposure was assessed by DiOC6 and flow cytometry. Subsequently lymphoma cells were exposed to itraconazole or vehicle and various chemotherapy agents such as doxorubicin (1µM), dexamethasone (1µM), cDDP (20μg/ml), bortezomib (20nM), carfilzomib (20nM) or MLN2238 (20nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Changes in hexokinase II (HKII), Voltage dependent anion channel protein (VDAC), LC3 and BCL-xL expression levels were determined by western blotting after exposure cells to itraconazole. VDAC-HKII interactions following in vitro exposure to itraconazole were determined by immunoprecipitation of VDAC and probing for HKII in RSCL and RRCLs. Result:Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition of all our sensitive and resistant lymphoma cell lines. In vitro exposure cells to itraconazole resulted in a loss of mitochondrial membrane potential and caused G2 cell cycle arrest. Itraconazole significantly had a synergistic anti-tumor effect combined with various chemotherapeutic agents, including doxorubicin, dexamethasone, cisplatin and different generations of proteasome inhibitors (bortezomib, carfilzomib or ixazomib) in both RSCL and RRCL. Western blot and immunoprecipitation studies demonstrated that following exposure to itraconazole, HKII bound less to mitochondrial specific protein VDAC. Complete silencing of HKII (using HKII siRNA interference) resulted in a rescue of loss in the mitochondrial membrane potential induced by intraconazole. Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The disruption of HKII from mitochondria following itraconazole exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic efficacy. Our finding highlights itraconazole as a potential therapeutic agent in the treatment of B-cell malignancies, and strongly supports clinical translation of its use. Disclosures No relevant conflicts of interest to declare.

The addition of rituximab to front line therapy for aggressive lymphomas has improved clinical outcomes, but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for rituximab associated chemotherapy cross-resistance our group developed several rituximab resistance cell lines (Raji 4RH and RL 4RH), which also display significant resistance to a wide range of chemotherapy agents. These rituximab resistant cell lines (RRCL)s exhibit multiple deregulations in the BCL-2 and inhibitor of apoptosis (IAP) protein families, including loss of the pro-apoptotic proteins Bax and Bak. We previously demonstrated that the X linked inhibitor of apoptosis protein (XIAP) is critically required for chemotherapy resistance in the RRCLs, and that an shRNA knockdown of XIAP increased chemotherapy response in both in vitro and in vivo models of rituximab resistant lymphoma. BMT-062789 is a heterodimeric mimetic of the second mitochondrial activator of caspases (SMAC) developed by Bristol-Myers Squibb, which can inhibit both the caspase 9 and caspase 3/7 binding domains of XIAP. BMT-062789 demonstrated dose and time dependent single agent anti-tumor effect (as measured by the Cell TiterGlo luminescent viability assay) in a panel of lymphoma cell lines, with IC50 values of less than 5uM for all cell lines tested except the rituximab resistant cell line Raji 4RH. The Burkitt's lymphoma cell line Daudi and diffuse large B-cell lymphoma cell line U2932 were particularly sensitive to BMT-062789 with IC50 values of 0.91uM and 0.76uM respectively. To investigate if the observed anti-tumor effect of BMT-062789 was due to increased apoptosis we exposed rituximab sensitive (Raji, RL) and rituximab resistant (Raji 4RH, RL 4RH) cells to escalating doses of BMT-062789 with or without the addition of 20uM etoposide for 48 hours. The induction of apoptosis was measured by flow cytometry with an Annexin-V:PE-Cy7 conjugate and Sytox blue (a DNA stain). 2uM BMT-062789 alone triggered apoptosis in 75% of Raji cells and 65% of RL cells. It is also worth adding that 2uM BMT-062789 induced higher rates of apoptosis than 20uM etoposide alone in both cell lines. The combination of 2uM BMT-062789 and 20uM etoposide together triggered apoptosis in 80% of Raji cells and 85% of RL cells, indicating that BMT-062789 may be able to augment the anti-tumor activity of conventional chemotherapy. More importantly, the combination of BMT-062789 and etoposide was also able to induce apoptosis in the rituximab resistant cell line models Raji 4RH and RL 4RH. 3uM BMT-062789 in combination with 20uM etoposide triggered apoptosis in 90% of Raji 4RH cells and 55% of the RL 4RH cells, which is a substantial improvement compared to 15% apoptosis with 20uM etoposide alone in each cell line. Additional studies are in progress to evaluate the anti-tumor effect of BMT-062789 in ex vivo samples from lymphoma patients with de novo and relapse/refractory disease. In summary, the novel heterodimeric XIAP inhibitor BMT-062789 has anti-tumor effect at low micromolar concentrations in lymphoma cell line models, including models of rituximab resistant disease. These results support earlier studies by our group indicating that XIAP is critical for survival in models of rituximab resistant lymphoma, and establish that XIAP inhibitors may have potential clinical value for the treatment of both de novo, and rituximab relapse/refractory lymphomas. Disclosures No relevant conflicts of interest to declare.

Targeting the proteasome system with bortezomib (BTZ) results in anti-tumour activity and potentiates the effects of chemotherapy/biological agents in multiple myeloma and B-cell lymphoma. Carfilzomib (CFZ) is a more selective proteasome inhibitor that is structurally distinct from BTZ. In an attempt to characterize its biological activity, we evaluated CFZ in several lymphoma pre-clinical models. Rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL), and primary tumour cells derived from B-cell lymphoma patients were exposed to CFZ or BTZ. Cell viability and changes in cell cycle were determined. Western blots were performed to detect PARP-cleavage and/or changes in Bcl-2 (BCL2) family members. CFZ was 10 times more active than BTZ and exhibited dose- and time-dependent cytotoxicity. CFZ exposure induced apoptosis by upregulation of Bak (BAK1) and subsequent PARP cleavage in RSCL and RRCL; it was also partially caspase-dependent. CFZ induced G2/M phase cell cycle arrest in RSCL. CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti-tumour activity of chemotherapy agents. Our data suggest that CFZ is able to overcome resistance to chemotherapeutic agents, upregulate pro-apoptotic proteins to promote apoptosis, and induce G2/M cell cycle arrest in lymphoma cells. Our pre-clinical data supports future clinical evaluation of CFZ in B-cell lymphoma.

Rituximab's primary mechanisms-of-action include: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CMC), induction of apoptosis and anti-proliferation. The acquirement of phenotypic changes in cancer cells or host immune cells over time may affect rituximab responsiveness and underscores the complexity of the potential mechanisms-of-resistance to anti-CD20 monoclonal antibodies (mAbs). Laboratory studies had demonstrated the importance of FcγRIIa and FcγRIII activating receptors in the biological activity of rituximab and other monoclonal antibodies (mAbs) in cancer medicine. Recently, investigators had focused their efforts in the study of FcγRIIb function expressed primarily on normal and malignant B-cells and its role in inflammatory conditions or hematological malignancies. FcγRIIb is an inhibitory low affinity Fcg receptor capable to induce potent inhibitory (apoptotic) signals via immune receptor tyrosine-based inhibition motif (ITIM). Previously, we generated several rituximab-resistance lymphoma cell lines (RRCL) and demonstrated a global down-regulation of CD20. On the other hand, forced expression of CD20 in RRCL did not restore rituximab sensitivity suggesting the existence of additional mechanisms of resistance to mAbs targeting CD20 (Tsai et al Clin Cancer Res. 2012;18:1039-50). To this end, we evaluated the expression and significance of FcγRIIb (CD32b) in RRCL. FcγRIIb gene and protein expression levels were determined in a panel of rituximab sensitive (RSCL) or RRCL by quantitative polymerase chain reaction (qPCR) and western blotting respectively. Subsequently, we silenced FcγRIIb in RSCL using siRNA interference and evaluated changes in the capacity of several mAbs targeting CD20 (rituximab, ofatumumab, and obinotuzumab) of inducing CMC or ADCC in vitro. RRCL were found to have almost a complete loss in FcgRIIb mRNA and protein levels when compared to RSCL. In addition, silencing of FcγRIIb in RSCL decreased the capacity of rituximab (P=0.04 [ADCC] and P=0.15 [CMC]), ofatumumab (P=0.02 [ADCC] and P =0.10 [CMC]), and obinituzumab (P=0.02 [ADCC] and P Disclosures No relevant conflicts of interest to declare.

Introduction: Metformin, a guanidine originally derived for Galega officinalis (French lilac), has been widely prescribed to type II diabetics since 1950 and well known for its safety toxicity profile. Recently, metformin use was associated with a decrease in risk cancer development and lower cancer-related mortality among breast, colorectal, prostrate, lung, hepatic and ovarian cancer patients. Our group previously reported that the use of metformin during front-line chemo-immunotherapy (i.e. R+CHOP) improved the clinical outcome of diffuse large B-cell lymphoma (DLBCL). The mechanism(s) of action underlying the antitumor effect of metformin remains not to be fully elucidated. Previous research done in our laboratory revealed that metformin inhibited cell proliferation through repressing PCNA and p21 proteins. Here, we report that metformin activates tumor suppressor p53 by suppressing MDMX in pre-clinical lymphoma models. Methods: A panel of rituximab-sensitive (RSCL), rituximab-resistant (RRCL) cell lines and primary tumor cells isolated from B-cell lymphoma patients were exposed to escalating doses of metformin (0-64mM). Changes in cell viability were determined by Presto Blue (Sigma) assay in cell lines and Titer Glo in patient samples. Changes in MDMX, MDM2 and p53 expression levels were determined by western blotting after exposure cells to metformin. MDMX-MDM2-p53 interactions and p53 ubiquitination following in vitro exposure to metformin were determined by immunoprecipitation of p53 and probing for MDMX, MDM2, ubiquitin and p53 in RSCL and RRCLs. Loss of Dψm or induction of apoptosis following metformin exposure were assessed by DiOC6 or Annexin V/PI staining and flow cytometry. Oxidative stress induced by metformin was measured by flow cytometry using dihydrorhodamine-123 (DHR-123) dye. Result: Metformin induced a dose-and time- dependent cell death in cell lines and primary patient tumor cells. In vitro exposure of lymphoma cell to metformin resulted in a decrease in MDMX levels. Immunoprecipitation studies demonstrated that following exposure to metformin, MDMX bound less to p53 leading to less p53 ubiquitination. In vitro exposure of RSCL or RRCL to metformin resulted in the expression of p53 regulated BH3 single domain proteins (Noxa and Puma). Moreover, metformin repressed mitochondrial potential, induced reactive oxidative species (ROS) generation and triggered apoptosis. Conclusion: Our data suggests that metformin had anti-tumor activity against RSCL, RRCL and primary tumor cells isolated from lymphoma patients. The down-regulation of MDMX and re-activation p53 function following metformin exposure may contribute to the disruption in the mitochondria potential, generation of ROS and induction of apoptosis observed in our models RSCL and RRCL. Our finding highlights a potential role for metformin in the treatment of B-cell malignancies. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Funds) Disclosures Czuczman: MorphoSys: Consultancy; Boehringer-Ingelheim: Other: Advisory Board; Celgene: Employment; Immunogen: Other: Advisory board.