Primary Mantle Cell Lymphoma Research Articles

Ironomycin Induces Mantle Cell Lymphoma Cell Death By Targeting Iron Metabolism Addiction

Mantle-cell lymphoma (MCL) accounts for approximately 5 to 7% of all lymphomas, and in North America and Europe. MCL remains an aggressive and incurable cancer with existing therapies, presenting a significant unmet clinical need. Accumulating evidence reveals that abnormal iron metabolism plays an important role in tumorigenesis and in cancer progression of many tumors. In cancer cells, the demand for iron increases in response to sustained deregulation of cell proliferation and DNA synthesis. Based on these data, we searched to identify alterations of iron homeostasis in MCL that could be exploited to develop novel therapeutic strategies. Analysis of the iron metabolism gene expression profile of a cohort of patients with MCL (n=71) established the iron score (IS), a gene expression-based risk score enabling identification of patients with MCL with a poor outcome who might benefit from a suitable targeted therapy. We analyzed the therapeutic interest of ironomycin, a new promising iron depleting molecule. Ironomycin is known to sequester iron in the lysosome and to induce ferroptosis (Mai TT et al. Nature Chemistry 2017). Preclinical studies in Acute Myeloid Leukemia (Garciaz et al., 2022 Cancer Discovery) and Diffuse Large B-Cell Lymphoma (Devin et al., 2022 Can Res) have revealed the therapeutic interest of ironomycin in treating hematological malignancies. In a panel of 6 MCL cell lines, ironomycin inhibited MCL cell growth at nanomolar concentrations compared with typical iron chelators. Then, we investigated if ironomycin induces cell death through apoptosis. Flow cytometry analysis revealed that ironomycin treatment resulted in AnnV+/7AAD+ and AnnV+/7AAD- death profiles in MCL. This effect was partially inhibited by the pan-caspase inhibitor Q-VD-Oph. Ironomycin also induced caspases 8 and 9 cleavage, and the activation of caspases 3/7. Furthermore, we found that ironomycin induces ferroptosis with lipid peroxidation and the production of ROS monitored by the fluorescence probe CM-H2DCFDA. Ironomycin induced cell death is inhibited by the ferroptosis inhibitor, ferrostatin-1. The treatment did not induce ferritinophagy, as there was no significant change on the expression of LC3B-II, ferritin and the transferrin receptor (CD71).Taken together, these data indicate that ironomycin kills MCL cells by apoptosis mediated by caspase activation and ferroptosis. Ironomycin significantly decreased cell proliferation rate, with a reduced percentage of cells in S-phase together with Ki67 and Cyclin D1 downregulation. Western blot analysis showed an increase of Chk1 and Chk2 phosphorylation, two central factors of DNA damage response, and an increase of the phosphorylation of γ-H2AX, indicative of DNA damage. With major importance, we validated the therapeutic interest of ironomycin in primary MCL cells of patients (n=5). Ironomycin (20 nM) demonstrated a significant higher toxicity in MCL cells compared to normal cells from the microenvironment. Furthermore, ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment. We tested the therapeutic interest to combine ironomycin with conventional chemotherapy used in MCL. Interestingly, we identified a synergistic effect when ironomycin is combined with Ibrutinib BTK inhibitor. Furthermore, Significant synergistic effects were also observed by combining ironomycin with BH3 mimetics or Doxorubicin. Altogether, these data underline that MCL patients my benefit from targeting iron homeostasis using ironomycin alone or in combination with conventional MCL treatments.

Abstract A02: Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981)

Abstract SUMOylation is a post-translational modification that leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. TAK-981, a novel selective inhibitor of SUMO-activating enzyme (SAEi) has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of non-Hodgkin lymphoma (NHL). TAK-981 was provided by Takeda (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL lines and led to rapid protein de-SUMOylation in a dose-dependent manner. TAK-981 induced pronounced DNA break in DLBCL cells (comet assay) and cell cycle arrest (propidium iodide). RNA-Seq and ATAC-Seq analysis of TAK-981-treated OCI-LY3 and U-2932 cells revealed upregulation of the NFκB, and OXPHOS signaling pathways. We noted that mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (EM) and a change of mitochondrial dynamics (ICC for p-Drp1), accompanied by rapid accumulation of reactive oxygen species. Seahorse respirometry revealed dramatic downmodulation of OXPHOS. Metabolomic profiling of DLBCL cells treated with TAK981 exhibited downmodulation of TCA substrates. Exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53 and DNA damage pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981 exposed splenocytes exhibited a reduction of maximal respiration by Seahorse. In summary, pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction and metabolic reprogramming. Citation Format: Tingting Liu, Vi Lam, Duanchen Sun, Elana Thieme, Tamilla Nechiporuk, Daniel Bottomly, Olga V Danilova, Nur Bruss, Martina Cusan, Lili Wang, Shannon K McWeeney, Jeffrey W Tyner, Steve Kurtz, Zheng Xia, Alexey Danilov. Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A02.

A Rare Case of Massive Bilateral Renomegaly due to Primary Involvement by Mantle Cell Lymphoma on 18F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography.

Mantle cell lymphoma (MCL) is a rare subtype of B-cell non-Hodgkin's lymphoma with an annual incidence of 5%, common in men with a median age of 60-70 years. Renal involvement is seen in about 0.7% of the extranodal lymphomas. The bilateral presentation is seen in 10%-20% of cases. There have been many case reports of MCL with renal involvement; however, primary renal involvement by MCL is extremely rare. We hereby describe the 18F-fluorodeoxyglucose positron emission tomography/computed tomographic findings in primary MCL involving the bilateral kidneys.

Abstract 3160: Establishment and characterization of a new mantle cell lymphoma cell line with a NOTCH2 mutation, ArBo

Abstract Cell lines represent a very useful tool for cancer research. In mantle cell lymphoma (MCL), the number of characterized cell lines remains small. We have recently established a novel blastoid MCL cell line, isolated from a malignant pleural effusion of a patient with MCL. A sample of the malignant pleural effusion was obtained with informed consent, and mononuclear cells were isolated. Following a few days of in-vitro culture, cells started to grow rapidly resulting in an estimated doubling time of 48h. The cells survived multiple freeze/thaw cycles and have been maintained in culture for the last 8 months. Hence the establishment of a novel blastoid MCL cell line, named Arbo. Both Arbo and the primary MCL cells obtained from the pleural effusion were negative for EBV DNA by qPCR. Flow cytometry showed kappa-restricted cells that were positive for CD5, 19, 20, 22, and 23, and negative for CD2, 3, 7, 10, and 71. FMC7 was positive in Arbo and negative in primary cells, as opposed to CD38 that was negative in Arbo and positive in the primary cells. Arbo’s karyotype was complex contrasting with the normal 46XY karyotype found in the primary MCL cells. The presence of t(11;14)(q13;q32) was confirmed by FISH, additionally overexpression of cyclin-D1 was confirmed by western blot. Whole exome sequencing (WES) was performed on DNA collected from Arbo revealing a total of 148,059 SNPs. Mutations were detected in the ATM, TP53 and NOTCH2(C19W and R2400*) genes. Mutations in NOTCH2 have not been described in other MCL cell lines, while they were identified in 5.2% of MCL patients. Immunoblotting for NOTCH2 confirms the expression of the protein by the cell line. Additionally, expression of CD23, which has been shown to be induced through NOTCH2 signaling, was confirmed by flow cytometry. To further characterize the effect of NOTCH2 on the survival of Arbo cells and CD23 expression, we cultured the cell line with increasing concentrations of the NOTCH2 transactivation inhibitor gliotoxin. We observed a dose-dependent inhibition of cell growth with an IC50 of 100 nM. Additionally, a dose dependent decrease in the expression of CD23 was observed by flow cytometry following treatment with gliotoxin. To determine the dependence of Arbo on B-cell receptor (BCR) signaling for survival, we cultured the cells with increasing concentrations of the BTK inhibitor ibrutinib for 48h. Inhibition of cell growth was observed in a dose-dependent manner resulting in an IC50 of 0.4 µM. In conclusion, Arbo is a novel blastoid MCL cell line with a NOTCH2 mutation that is fully characterized and will be made available to the research community. Supported by a grant from the Ladies Leukemia League, Inc., of the Gulf South Region. Citation Format: Ishwarya Satyavarapu, Firas M. Safa, Terri Rasmussen, Nakhle Saba. Establishment and characterization of a new mantle cell lymphoma cell line with a NOTCH2 mutation, ArBo [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 3160.

Abstract 199: A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments

Abstract Mantle cell lymphomas (MCL) are rare B-cell neoplasms belonging to the non-Hodgkin’s lymphomas and often show an aggressive clinical course. MCL respond relatively briefly to conventional chemotherapy and are characterized by frequent relapses (Dreyling et al., Ann Oncol, 2017). Therapy for MCL remains a challenge also due to limited cultivation time of primary cells. In vitro suspension culture of primary human MCL cells is not easily feasible (Bryant et al., Lab Invest, 2000) and cells undergo spontaneous apoptosis ex vivo (Zhang et al., Blood, 2012; Medina et al., Heamatologica, 2012). Useful in vitro MCL models that allow long-term culture of primary MCL cells could be helpful in the biological and therapeutic study of MCL. For this purpose, we established a 3D hydrogel culture model for cultivation of primary MCL cells that were first tested with cell lines. After 3 days of culture, the cells cultured in the hydrogel can successfully be analysed for cell viability (live-dead confocal laser scanning microscope-based assay, flow cytometry), metabolism (pH, glucose, lactate), and biomarker expression by immunohistochemistry. The 3D hydrogel cultivation model allows a variety of analytical methods, also because it is reversible and the cells can easily be separated as single cell suspension from hydrogel for further analytical purposes, such as flow cytometric analyses. Next, we cultured primary MCL cells in the alginate hydrogel 3D culture system. MCL cells were isolated from blood of different patients and separated by Ficoll centrifugation. The isolated cell population was identified as MCL cells by double-staining of anti-CD20 and anti-CD5 followed by flow cytometric analysis. Cells were further cultured in medium as conventional suspension culture or in alginate hydrogel as 3D culture. Cell death was assessed with AnnexinV by staining phospholipids of apoptotic cells, allowing flow cytometric quantification. All samples had less than 6% AnnexinV positive (+) cells at day 0 before the experiment started. After 2 days cultivation, primary MCL cells showed a strong induction of cell death in suspension culture with 65.5% (±4.2%) AnnexinV+ cells versus 21.9% (±0.6%) AnnexinV+ cells in 3D hydrogel culture. Subsequently after 3 days cultivation, 74.2% (±12.2%) AnnexinV+ cells in suspension versus 33.5% (±1.3%) AnnexinV+ cells in the hydrogel 3D culture were determined. Therefore, we conclude that our novel 3D culture system might be a more suitable cultivation method for primary MCL cells than conventional suspension culture in cell culture medium. We have developed a 3D cultivation system with alginate hydrogel that allows nearly tripling the in vitro cultivation time of primary MCL. This could pave the way for new long-term treatments in research in the future and serve a better understanding of the cells. Citation Format: Kathrin Böpple, Annette M. Staiger, Heike Horn, German Ott, Walter E. Aulitzky, Meng Dong. A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments [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 199.

Abstract 6327: Profiling T-cell subpopulations based on spatial proteome in mantle cell lymphoma

Abstract Background: In precision medicine era, we need to develop tools that better predict treatment response with, for e.g. immuno-oncology drugs. One hypothesis is that diverse treatment response is governed by differences in the tumor microenvironment (TME) both within the tumor and surrounding areas. Thus, novel ways to measure the heterogeneity within defined immune-cell populations is the key and now possible by using spatially-resolved multiplexed platforms. Here, we have investigated the phenotype of four T-cell subtypes both within and excluded from the tumor-rich regions in a large cohort of primary mantle cell lymphoma (MCL) patients. We propose that profiling different cell populations could potentially provide insight into patient prognosis and identify clinically actionable targets, but also in understanding whether tumor cells locally re-educate T-cells through cell-to-cell interaction or by soluble factors Method: We analyzed archival FFPE presented as tissue microarrays of 196 cores from 104 MCL patients, collected from the Biobank of Lymphoma of Southern Sweden, by using the, GeoMX-digital spatial profiler (DSP, Nanostring Inc.). Cellular regions of interest were selected by immunofluorescense staining of CD8, CD3 and CD57 to identify four subpopulations of T cells - early cytotoxic (CD3+ CD8+ CD57-), early non-cytotoxic (CD3+ CD8- CD57-), late cytotoxic (CD3+ CD8+ CD57+) and late non-cytotoxic (CD3+ CD8- CD57+). 601 regions distributed among the four T-cell subtypes were collected from both within the tumor-rich and tumor deficient zones and probed for 69 proteins for sub-characterization. Result: Our results show that the distribution and expression profile of all subpopulations differ considerably based on their position relative to the tumor. Using linear mixed effects model to account for effect of multiple sampling per patient, we observed higher cytolytic activity (upregulation of granzyme A and B) (FDR cut-off < 0.05) in early cytotoxic and late non-cytotoxic cells, within the tumor-rich zone compared to the tumor-deficient area. In contrast, proteins such as STING, VISTA, neurofibromin and fibronectin were consecutively upregulated in all subtypes in tumor-deficient zones. We also observe differential expression profiles for proteins involved in for example cell death regulation and apoptotic signaling pathways. Our analysis provide insight into effector functions important for response to treatment in MCL. Conclusion: This study reveals the phenotypic differences of T-cell subsets in MCL with a higher resolution compared to previous studies. The spatially-resolved analysis shows that the proximity to tumor cells highly affect the profile of different T-cell subsets and that further analysis of re-education mechanisms should be pursued. This study advance the understanding of the MCL tissue microenvironment and for the basis for connecting such differences to clinical outcome. Citation Format: Lavanya Lokhande, Joana de Matos Rodrigues, Anna Sandstrom Gerdtsson, Anna Porwit, Mats Jerkeman, Sara Ek. Profiling T-cell subpopulations based on spatial proteome in mantle cell lymphoma [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 6327.

CD24 Is a Potential Immunotherapeutic Target for Mantle Cell Lymphoma.

CD24 and its ligand Siglec-10 were described as an innate immune checkpoint in carcinoma. Here, we investigated this axis in B-cell lymphoma by assessing CD24 expression and evaluating pro-phagocytic effects of CD24 antibody treatment in comparison to hallmark immune checkpoint CD47. In mantle cell lymphoma (MCL) and follicular lymphoma patients, high mRNA expression of CD24 correlated with poor overall survival, whereas CD47 expression did not. Conversely, CD24 expression did not correlate with survival in diffuse large B-cell lymphoma (DLBCL), whereas CD47 did. CD24 was also highly expressed on MCL cell lines, where treatment with CD24 antibody clones SN3 or ML5 potently induced phagocytosis, with SN3 yielding >90% removal of MCL cells and triggering phagocytosis of primary patient-derived MCL cells by autologous macrophages. Treatment with CD24 mAb was superior to CD47 mAb in MCL and was comparable in magnitude to the effect observed in carcinoma lines. Reversely, CD24 mAb treatment was less effective than CD47 mAb treatment in DLBCL. Finally, phagocytic activity of clone SN3 appeared at least partly independent of antibody-dependent cellular phagocytosis (ADCP), suggesting CD24/Siglec-10 checkpoint activity, whereas clone ML5 solely induced ADCP. In conclusion, CD24 is an immunotherapeutic target of potential clinical relevance for MCL, but not DLBCL.

Specific location of ocular adnexal lymphoma and mortality: an international multicentre retrospective study

AimsTo examine whether the specific location of ocular adnexal lymphoma (OAL) and the American Joint Committee on Cancer (AJCC) TNM tumour stage are prognostic factors for mortality in the main...

The IL32/BAFF axis supports prosurvival dialogs in the lymphoma ecosystem and is disrupted by NIK inhibition.

Aggressive B-cell malignancies, such as mantle cell lymphoma (MCL), are microenvironment-dependent tumors and a better understanding of the dialogs occurring in lymphoma-protective ecosystems will provide new perspectives to increase treatment efficiency. To identify novel molecular regulations, we performed a transcriptomic analysis based on the comparison of circulating MCL cells (n=77) versus MCL lymph nodes (n=107) together with RNA sequencing of malignant (n=8) versus normal B-cell (n=6) samples. This integrated analysis led to the discovery of microenvironment-dependent and tumor-specific secretion of interleukin-32 beta (IL32β), whose expression was confirmed in situ within MCL lymph nodes by multiplex immunohistochemistry. Using ex vivo models of primary MCL cells (n=23), we demonstrated that, through the secretion of IL32β, the tumor was able to polarize monocytes into specific MCL-associated macrophages, which in turn favor tumor survival. We highlighted that while IL32β-stimulated macrophages secreted several protumoral factors, they supported tumor survival through a soluble dialog, mostly driven by BAFF. Finally, we demonstrated the efficacy of selective NIK/alternative-NFkB inhibition to counteract microenvironment-dependent induction of IL32β and BAFF-dependent survival of MCL cells. These data uncovered the IL32β/BAFF axis as a previously undescribed pathway involved in lymphoma-associated macrophage polarization and tumor survival, which could be counteracted through selective NIK inhibition.

Dual BTK/SYK inhibition with CG-806 (luxeptinib) disrupts B-cell receptor and Bcl-2 signaling networks in mantle cell lymphoma

Aberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.

Primary Mantle Cell Lymphoma of Pancreas- Rare Case

Primary pancreatic lymphomas (PPLs) are rare cancers that have a complicated histological classification. Sufficient sample volumes are required to adequately diagnose and decide the best treatment for PPLs. A 54-year-old man with primary pancreatic mantle cell lymphoma is described in this case study. Percutaneous biopsy was used to extract histological samples. Atypical small to medium round cells predominated in the tumor cells, which had diffuse CD20 and cyclin D1 positivity. Chemotherapy was used on the patient, and he went into complete remission as a result. Because it provides adequate samples for testing, a percutaneous biopsy could be a valuable and safe diagnostic tool for PPLs.

A nodular type of mantle cell lymphoma in the nasopharynx.

Mantle cell lymphoma (MCL) is a subtype of B-cell lymphoma derived from CD5-positive antigen-naïve pre-germinal center B-cells within the mantle zone that surrounds normal germinal center follicles. MCL comprises approximately 5 to 10% of all lymphomas. Tonsil is the most common location of MCL in the head and neck region, followed by the nasopharynx. Primary MCL involving the nasopharynx is extremely rare. Its clinical course is very aggressive with frequent relapses after conventional chemotherapy. It always presents as a protruding mass on the mucosal lining of the pharyngeal cavity. Here, we report a new nodular type of MCL in the nasopharynx. Endoscopically, this case showed multiple nodular lesions of primary MCL on the nasopharyngeal mucosa. This unique finding has not been reported yet in the English literature. These lesions should be differentiated from simple pharyngeal infections or benign lymphoid hyperplasia in the nasopharynx.

Epidermal Growth Factor-like 7 As a Novel Therapeutic Target in Mantle Cell Lymphoma

▪Mantle cell lymphoma (MCL) is an aggressive form of mature B-cell non-Hodgkin's lymphoma (NHL), accounting for nearly 6% of NHL cases. Currently, MCL patients are treated with aggressive chemo-immunotherapy regimens followed mostly by consolidation with autologous stem cell transplantation and maintenance rituximab. Despite these intensive therapies, MCL prognosis remains poor, with a median overall survival of 6-7 years, with most of the patients developing the refractory or recurrent disease. Thus, there is a need for novel and more effective, less toxic therapies for MCL. Epidermal growth factor-like 7 (EGFL7) is a protein secreted by endothelial cells and plays a critical role in angiogenesis. Our lab was the first to demonstrate a role for EGFL7 in hematologic malignancies, demonstrating that EGFL7 is increased in leukemic blasts of AML patients and that anti-EGFL7 treatment alone results in prolonged survival of leukemic mice (Papaioannou et al., 2017). While EGFL7 has been shown to play a role in some hematological malignancies, its role in MCL has not been investigated. Therefore, we assessed EGFL7 expression levels in MCL patients compared to healthy controls using the publicly available dataset GSE46846. We found significant increases in EGFL7 in malignant B cells from MCL patients compared to healthy individuals (p<0.05). Furthermore, using the publicly available clinical data set (Scott et al., 2017), we found that MCL patients (n=122) with high EGFL7 expression associated with lower overall survival rates compared to MCL patients with low EGFL7 (24 months; vs. 48 months, respectively, p= 0.0057) (Figure 1).To examine the therapeutic potential of targeting EGFL7 in MCL cells, we treated patient-derived xenograft (PDX) cells (n=3) with an anti-EGFL7 blocking antibody (Parsatuzumab) in vitro. We found an increase in apoptosis of MCL PDX cells compared to IgG control (15-50% vs. 0.5-2.4%, respectively), p<0.0001. Similar results were found when treating three MCL cell lines (Rec1, Jeko1, and SP53) with anti-EGFL7 or control. We found a decrease in cell proliferation (20 vs. 70%, p<0.0001) and an increase in apoptosis (67-87% vs. 8-17%, p<0.0001) at 48-hours post-anti-EGFL7 treatment compared to IgG, respectively. Next, to determine whether anti-EGFL7 treatment could target MCL cells in vivo, NSG mice were subcutaneously injected with Rec1 cells (5 x10 6). Seven days post-injection, mice were treated with anti-EGFL7 or IgG (50mg/kg, three times/week) (n=5 per group). Tumors were measured every week, and mice were sacrificed when they reached end point criteria. We found that anti-EGFL7 treated mice had significantly decreased tumor volume than IgG (1116.58mm 3 vs. 3626mm 3, respectively, p=0.0116) and increased survival (p = 0.0034). Overall, our data show that targeting EGFL7 using an anti-EGFL7 blocking antibody inhibits MCL cell growth and prolongs survival in mouse models of MCL.Our lab has previously shown that EGFL7 binds to the Epidermal growth factor receptor (EGFR) in AML (Bill et al., 2020). Knowing the importance of EGFR in lymphoma, we validated the binding of EGFL7 to EGFR in MCL cells by performing an immunoprecipitation (IP) assay on protein lysates from PDX cells (n=2) and Jeko1 cells. We found that EGFL7 protein was significantly enriched in protein fractions pulled down using anti-EGFR antibody compared to IgG. Conversely, we transfected Jeko1 cells with Flag-tagged EGFL7 plasmid and performed IP using anti-Flag antibody. EGFR protein was significantly enriched in the protein fraction pulled down using an anti-Flag antibody compared to IgG. Next, we examined the association between EGFL7 and EGFR expression in primary MCL patients and found that EGFR positively correlates with EGFL7 expression (n=122, r=0.1533). Further, Anti-EGFL7 treatment decreased phospho-AKT protein levels in PDX cells and MCL cell lines compared to IgG control, suggesting blocking EGFL7 abrogates EGFR mediated downstream signals.In conclusion, this is the first report describing a role for EGFL7 in MCL growth and/or survival by modulating the EGFR-AKT signaling pathway and targeting EGFL7 using an anti-EGFL7 blocking antibody as a novel treatment to improve the outcome for MCL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Novel Multitarget Small-Molecule Inhibitor SRX3177 Overcomes Ibrutinib Resistance in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive, rare, and difficult to treat subtype of non-Hodgkin's Lymphoma (NHL) that accounts for about 6% of all cases. Although there is no defined standard of care for MCL treatment, some combination of chemo-immunotherapy and rituximab maintenance with or without autologous stem cell transplantation is generally employed depending on the age and fitness of the patient. Despite recent development of novel therapeutics, there is inevitable disease relapse with progressively declining efficacy and increasing frequency of resistance with single agent targeted therapy. Here, we describe the novel multitarget inhibitor SRX3177 which simultaneously hits three oncogenic targets: phosphatidylinositol-3 kinase (PI3K), cyclin-dependent kinases 4 and 6 (CDK4/6), and the epigenetic reader protein BRD4. This in silico designed, thieno-pyranone (TP) scaffold-based small molecule inhibitor orthogonally disrupt three targets within the cancer cell with one agent.Targeting the cell cycle with small molecule inhibitors represents a reasonable attempt to treat MCL, as cell cycle-associated genes like ATM, TP53, CDKN2A, CCND1 and CDK4/6 are most frequently mutated in patients. Palbociclib is a well-known single agent CDK4/6 inhibitor that has been employed in both solid and hematological malignancies. Due to its cytostatic nature, treatment with single agent palbociclib often results in the emergence of treatment-resistant clones. Therefore, a combination strategy would theoretically be more effective and can overcome the development of resistance. Moreover, prolonged G1 arrest by CDK4/6 inhibition sensitizes lymphoma cells to PI3K inhibition, suggesting a synthetic lethality relationship between these two agents. Inhibiting the chromatin reader protein BRD4 causes downregulation of target genes c-MYC and BCL2, further increasing cytotoxic capabilities. Hence, we developed SRX3177 as a potent CDK4/6/PI3K/BRD4 triple inhibitor to synergistically inhibit cell cycle progression and induce cancer cell apoptosis.SRX3177 is an ATP competitive CDK4/6 inhibitor (IC 50: CDK4 = 2.54 nM, CDK6 = 3.26 nM), PI3K inhibitor (IC 50: PI3Kα = 79.3 nM, PI3Kδ = 83.4 nM), and BRD4 inhibitor (IC 50: BD1 = 32.9 nM, BD2 = 88.8 nM). We have tested the efficacy of SRX3177 against a panel of MCL cell lines and report that SRX3177 induces a strong antiproliferative activity with maximal IC 50 0f 340 nM in JeKo-1, 29 nM in Mino cells, and 630 nM for Rec-1 cells while IC 50 values for cell lines Granta and JVM-2 were 1.3 µM and 1.5 µM, respectively. Further, we show that SRX3177 is more potent to tumor cells than the individual PI3K (BKM120), BTK (Ibrutinib), BRD4 (JQ1), and CDK4/6 (palbociclib) inhibitors, and dual PI3K/BRD4 inhibitor SF2523 (backbone for SRX3177) in JeKo-1 cells. Next, we examine the cytotoxic effect of SRX3177 in ibrutinib/palbociclib resistant primary MCL cells. Our results show that SRX3177 triggers cytotoxic response at 500 nM and 1000 nM as compared to the lack of cytotoxicity of combination Ibrutinib and palbociclib at 150 nM and 1000 nM (Fig 1). SRX3177 induces a strong apoptotic response and cell cycle arrest in JeKo-1 and Mino cells at 24hrs. Annexin V/7AAD apoptosis staining confirmed the induction of PCD by SRX3177with increase in c-PARP. Western blot analysis shows SRX3177 treatment blocks both PI3K/AKT signaling and Rb phosphorylation. Moreover, analysis by chromatin immunoprecipitation revealed that SRX3177 effectively blocked BRD4 binding to both the promoter and enhancer of c-MYC (p≤0.01 and p≤0.001) and BCL2 (p≤0.05). SRX3177 also suppresses the c-MYC and BCL2 transcriptional program in both a time- and dose-dependent manner. Our findings also demonstrate a SRX3177-dependent reduction in c-MYC half-life via induction of proteasomal-mediated degradation. This degradation is associated with decreased phosphorylation of c-MYC at Ser62 and increased phosphorylation of c-MYC at Thr58 - indicative of differential regulation of c-MYC stability. Finally, we show that SRX3177 overcomes chronic ibrutinib resistance in Jeko-1 cells with a maximal IC 50 of 150 nM as compared to 64 µM with ibrutinib. Hence, the triple inhibitor SRX3177 has superior potency to ibrutinib in MCL cell lines and succeeds in overcoming ibrutinib-resistance at nanomolar doses. Taken together, our data supports the development of SRX3177 as a novel therapeutic agent for treatment of MCL. [Display omitted] DisclosuresMartin: ADCT: Consultancy. Park: Takeda: Research Funding; G1 Therapeutics: Consultancy; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding, Speakers Bureau; Gilead: Speakers Bureau; Rafael Pharma: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Morphosys: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding. Durden: SignalRx Pharmaceuticals: Current holder of individual stocks in a privately-held company.

The IL32/BAFF Axis Supports Prosurvival Dialog in the Lymphoma Ecosystem and Is Disrupted By NIK Inhibition

BackgroundAggressive B-cell lymphomas, such as Mantle cell lymphoma (MCL), are microenvironment-dependent tumors but, in contrast to tumoral intrinsic anomalies, complex interplays within their ecosystems are largely ignored. A better understanding of these dialogs could provide new perspectives integrating the key role of the microenvironment to increase treatment efficiency of this hard to cure B-cell malignancy.MethodsTo identify novel molecular regulations occurring in lymphoma protective ecosystems, we performed a transcriptomic analysis based on the comparison of publicly available datasets from circulating (PB, n=77) versus MCL lymph nodes (LN, n=107) together with deep RNA sequencing of purified CD19+CD5+ MCL (n=8) versus normal B-cells (n=6). This integrated analysis led to the discovery of microenvironment-dependent and tumor-specific secretion of the cytokine IL32β by lymphoma cells. Using in situ multiplex immunohistochemistry , ex vivo models of primary MCL cells (n=23) and IL32 KO MCL cell lines (Crispr/Cas9), we studied the regulation and the functional impact of IL32β in the MCL context, especially in the dialog with tumor-associated macrophages.ResultsAmong the 6887 genes differentially expressed in MCL LN compared to PB in vivo, 70% were confirmed in CD19+ MCL cells cocultured ex vivo and 39% were tumor-specific, that is to say not upregulated in cocultured normal B cells (NBC). Top-genes scoring revealed that IL32 was the most upregulated genes within the “Tumor-specific” transcriptional program. Using ex vivo models of primary MCL cells, we demonstrated that the microenvironment-dependent secretion of IL32β was controlled by the CD40/NFKB2 axis whereas its tumor specificity was the consequence of IL32 promoter hypomethylation in MCL compared to NBC.IL32 protein expression was confirmed in MCL LN in situ by multiplex IHC. The latter allowed the concurrent detection of MCL cells, T cells, macrophages and IL32. Consistently with the microenvironment-dependent induction of IL32 in MCL, we observed that IL32 expression was enriched in situ in tumor zones infiltrated with T cells, compared to tumor-exclusive zones.Based on in vitro experiments using IL32 KO MINO cells (Crispr/Cas9), we demonstrated that, through the secretion of IL32β, the tumor was able to corrupt its microenvironment by polarizing monocytes into specific protumoral CD163 mid MCL-associated macrophages, which secrete both pro- (e.g. IL6, OSM, IL1a/b) and anti- inflammatory (e.g. IL10, IDO, IL18, IL4L1) soluble factors. We next highlighted that IL32β-stimulated macrophages supported tumor survival mostly through a soluble dialog, which is driven by BAFF.Finally, we demonstrated the efficacy of selective NIK/alternative-NFkB inhibition to counteract both microenvironment-dependent induction of IL32β (RNA expression inhibition: 62 % ; n= 3) and BAFF-dependent survival of MCL cells (survival support inhibition : 47 % ; n=6).ConclusionsIn summary, our data uncovered the IL32β/BAFF axis as a previously undescribed pathway involved in MCL-associated macrophage polarization and tumor survival. Dependent on alternative-NFkB signaling, tumor-specific secretion of IL32β led to the corruption of the microenvironment through the polarization of monocytes into specific MCL-associated macrophages, which in turn favor tumor survival. While IL32β-stimulated macrophages secreted several protumoral factors, they supported MCL survival through BAFF and consequent alternative-NFkB activation in tumor cells. Our data shows that targeting IL32b, BAFF or the alternative NFkB pathway through NIK inhibition could also be of major interest for counteracting the multiple cross-talks that occur in the MCL microenvironment and, especially, the CD40L + T-cell / MCL / CD163 + MCL-associated macrophage triad. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bispecific CD19-CD20 and CD19-CD22 CAR-T Cells with Glycogen Synthase Kinase (GSK)-3β Inhibitor TWS119 Treatment Have Superior Therapeutic Effects on Mantle Cell Lymphoma

Introduction: Chimeric antigen receptor (CAR) - expressing T cells have shown breakthrough clinical successes to hematological malignancies such as diffuse large B cell lymphoma. Intensive development on CAR-T cell therapies are being developed in many common lymphomas. However, the development of CAR-T cell therapies in mantle cell lymphoma (MCL), a rare lymphoma has lagged behind. So far only CD19 CAR-T has been applied to MCL treatment, and MCL frequent relapse from prior CD19 CAR-T therapy with CD19 antigen loss. Bispecific CAR-T cells target two tumor antigens at once could reduce the risk of relapse. In addition, immune checkpoint blockade such as GSK3β and PD-1inhibition has been successfully used to enhance CAR-T cell therapies. Therefore, we attempt to develop a dual CAR for mantle cell lymphoma with checkpoint blockade.Methods: CD19-CD20 and CD19-CD22 bispecific CAR-T constructs were generated by subcloning the fragments in-frame into the pSFG retroviral vector. The BiCAR-T cells were cultured with/without a small molecule GSK3β inhibitor TWS119. Then, we performed a serial of experiments using CARs. 1, We assayed the CAR expression and cell expansion rates in vitro. 2, We assessed the tumor-killing capacity in vitro using MCL cell lines and primary MCL patient samples. 3, We performed phenotypic analyses by determination of proteins expression profiles of exhaustion markers and stem cell markers by flow cytometry. 4, We examined in vivo antitumor efficacy using preclinical models of MCL cell line-derived xenograft (CDX) .Results: Firstly, we found CD19-CD20 and CD19-CD22 bispecific CAR-T cells showed similar CAR expression levels (55%-72% CAR+ ) and similar expansion rate as single CD19 or CD20 CAR. Secondly, bispecific CAR- T cells potently and specifically lysed tumor cells in vitro, and showed the better killing of MCL tumor than CD19 or CD20 single CAR after challenge with MCL cell lines in vitro. Finally, we also confirmed these effects in CDX mice models derived from MCL cell line, Jeko-1 in vivo. CD19-CD20 and CD19-CD22 bispecific CAR successfully eliminated MCL tumor, showed better antitumor efficacy, and prolonged mouse survival than single CAR treated groups (P < 0.001). We also found that CD19-CD20 and CD19-CD22 bispecific-CAR T cells treated with TWS119 express significantly fewer exhaustion markers, such as PD-1 (P < 0.001), and LAG3 (P < 0.001), and showed better antitumor efficacy than the nontreated control group and single CAR groups in vivo. Furthermore, CAR-T cells expressing bispecific CD19-CD20 and CD19-CD22 CAR with TWS119 treatment had superior anti-MCL activity in vitro. In total, significantly better anti-MCL effects were observed for CD19-CD20 and CD19-CD22 bispecific CARs than single CAR. Their anti-MCL functions were further improved by TWS119 treatment.Conclusion: Altogether, these findings suggest that bispecific CD19-CD20 and CD19-CD22 CAR-T cells are potent for treating MCL relapse and drug resistant patients. In addition, bispecific CD19-CD20 and CD19-CD22 CAR-T cells with TWS119 treatment will further bring up the full power of CARs. These data support the therapeutic potential of bispecific CARs in patients with MCL. DisclosuresWang: Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; BGICS: Honoraria; Bayer Healthcare: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Clinical Care Options: Honoraria; Lilly: Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Mumbai Hematology Group: Honoraria; Epizyme: Consultancy, Honoraria; Dava Oncology: Honoraria; Chinese Medical Association: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Molecular Templates: Research Funding; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; VelosBio: Consultancy, Research Funding; InnoCare: Consultancy, Research Funding; Genentech: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; CStone: Consultancy; Scripps: Honoraria; OMI: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Oncternal: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Anticancer Association: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding.

A Unique Case of Mantle Cell Lymphoma Masquerading as a Cecal Mass

Mantle cell lymphoma (MCL), a type of B-cell non-Hodgkin's lymphoma, is a rare and aggressive disease with a poor prognosis due to its advanced presentation at diagnosis. It is characterized by a translocation in the Bcl-1 gene, which results in overexpression of cyclin D1. MCL is frequently seen in the form of multiple lymphomatous polyposis (MLP) in which innumerable polyps are observed in the gastrointestinal (GI) tract. In rare instances, MCL presents a single mass. The most common presentation involves male patients in their sixties, with generalized lymphadenopathy, extranodal involvement, and B symptoms (night sweats, fever, and weight loss). Endoscopic findings of MLP include cerebroid folding of the gastric mucosa and innumerable polyps extending from the duodenum to the large intestine and are reported in approximately 9% of all GI lymphomas. Less commonly, only 2–4% of GI malignancies present as a primary GI MCL as a single mass, usually in the stomach and ileocecal region in the intestine. Radiologic findings include lymphadenopathy, splenomegaly, multiple polyposis, or wall thickening with ulceration or mass formation. In most instances, advanced disease is found at diagnosis, for which 5-year survival ranges only from 26 to 46%, even when appropriate treatment is initiated. High mitotic rate, or Ki-67 index, is of prognostic value and is associated with poor prognosis. Treatment involves conventional chemo-immunotherapy consisting of R CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) or RB (rituximab and bendamustine), with the latter being better tolerated and associated with longer progression-free survival. Surgical resection is usually limited to patients in which complications are seen such as bleeding, perforation, or bowel obstruction. We present a unique case of a 70-year-old male with nonbilious, nonbloody emesis, and symptomatic anemia who was found to have a cecal mass consistent with MCL.

Symptomatic Thyroglossal Duct Cyst Presenting With Synchronous and Localized Tongue Base Extranodal Mantle Cell Lymphoma. A Unique Case Report Demonstrating Lymphoma Colonization of Cyst Wall, Potentially Relating to a Persistent Embryological Foramen Cecum Remnant

A 77-year-old male presented with a progressively enlarging midline neck mass. On further investigation he was found to have synchronous thyroglossal duct cyst and extranodal mantle cell lymphoma (MCL) localized to the base of tongue. Both pathologies were managed simultaneously with a surgical approach and the patient remained in clinical remission at the time of publication without indication for systemic oncological treatment. Histology revealed primary extranodal nonblastoid MCL forming a base of tongue mass, with colonization of the thyroglossal duct cyst. Lymphoma was also found in the epithelium of a crypt-like tract traversing one of the tongue base tumor sections. This tract was anatomically and histologically consistent with documented descriptions of the foramen cecum. This case report illustrates a previously undescribed temporal, clinical, and histological association between a base of tongue MCL and symptomatic thyroglossal duct cyst. We provide evidence for a potential causal relationship for the presentation of the thyroglossal duct cyst as a result of oropharyngeal MCL, in the absence of clinical and histological evidence of disseminated disease, directly infiltrating from its tongue base origin to the infrahyoid neck region, potentially via an embryologic foramen cecum remnant. We also highlight the crucial role of the histopathologist in multidisciplinary clinicopathological discussion in demonstrating how fundamental embryological and microanatomical relationships can unite apparently separate diseases.

Drug Screening Reveals That Ibrutinib (PCI-32765) Exhibits Synergy With BCL-2 and Proteasome Inhibitors In Models Of Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL)

BackgroundIbrutinib, a Bruton's Tyrosine Kinase inhibitor, is clinically effective in relapsed MCL and CLL, but responses are often partial and resistance eventually develops. To identify agents that synergize with ibrutinib, we performed an in vitro combinatorial drug screen and evaluated the most promising combinations in additional models. MethodsDrug screens were performed in JVM2 and Z138 MCL cell lines, combining ibrutinib with a diverse panel of secondary agents inhibiting targets within the B-cell receptor (BCR) pathway (SC514, PD325901, enzastaurin, R788, temsirolimus, dasatinib, idelalisib) and agents inhibiting targets outside the BCR pathway (ABT-199, bendamustine, carfilzomib, bortezomib, geldanamycin, panobinostat, PD0332991). Three doses (IC10, IC20, IC 30) for each drug were chosen and combined in a 3x3 format. Bliss Model of Additivity was used to quantitate synergistic cytotoxicity. Levels of cleaved PARP or Annexin V/PI staining were assessed for cell lines exposed to ibrutinib and synergistic agents, including bortezomib, carfilzomib, and ABT-199. The Ibrutinib + carfilzomib combination was further tested in MCL cell lines Rec1 and Jeko, in primary MCL and CLL patient (pt) samples, and in a MCL-SCID-hu mouse. Molecular analysis of the mechanistic interactions of the agents has been initiated using forward-phase antibody arrays (R and D Systems). ResultsInhibitors of targets within the BCR pathway were ineffective at potentiating the effects of ibrutinib (magnitude of synergy ranging -12%-9% in Z138, -25%-8% in JVM2). However, substantial synergistic cytotoxicity was seen with some agents that target outside the BCR pathway (magnitude of synergy ranging -5%-37% in Z138, -17%-48% in JVM2). Notably, we observed robust and reproducible synergistic cytotoxicity by combining ibrutinib with proteasome inhibitors carfilzomib and bortezomib as well as BCL-2 inhibitor ABT-199.The degree of synergyof ibrutinib + ABT-199 in MCL cell lines was durable across all 9 dosing combinations, (23-32% in Z-138, 10-26% in JVM-2). In CLL pt samples, ibrutinib + ABT-199 conferred modest synergy (10-20%) and near-complete cytotoxicity (61-70%). Flow cytometry for cleaved PARP demonstrated a high degree of apoptosis when combining ibrutinib with ABT-199 (94.5% in Z-138, 82.1% in Z-138).At optimal dosing, bortezomib conferred 19% synergy with ibrutinib in Z138 and 27% in JVM2. The best synergistic effect of carfilzomib and ibrutinib was 50% in Z138 and 65% in JVM2. The synergistic effect between carfilzomib and ibrutinib was maintained in Rec1 and Jeko MCL cell lines and in primary MCL pt samples. In CLL pt samples, ibrutinib + proteasome inhibition also conferred high degrees of total cytotoxicity (58-71% for carfilzomib, 42-62% for bortezomib). Flow cytometry for cleaved PARP or Annexin V/PI demonstrated increased apoptosis for ibrutinib +carfilzomib (92.3% in Z138, 61.2% in JVM2, 95% in Rec1, 90% in Jeko). The combination treatment increased the percent survival of MCL-SCID-hu mice in three-fold over single agent treatment in a 125-day trial.Preliminary analysis of proteomic changes induced by ibrutinib+ carfilzomib indicates synergistic alterations in cell cycle proteins and in apoptosis-regulating proteins, although the specific alterations differ between Z138 and JVM2 cell lines. DiscussionWhile agents within the BCR pathway did not display synergistic cytotoxicity with ibrutinib in our drug screen, several agents outside the BCR pathway did synergize with ibrutinib, notably ABT-199, bortezomib, and carfilzomib in two MCL cell lines. For all three drug combinations, this effect appeared to be mediated via apoptosis. The synergistic effect of ibrutinib + carfilzomib was further studied in 2 additional MCL cell lines, 3 MCL pt samples, and CLL pt samples. MCL-SCID-hu mice demonstrated three-fold increased survival when treated in combination over animals treated with either agent alone. The identification of unexpected synergistic cytotoxic interactions between inhibitors of apparently unrelated pathways is a benefit of our combinatorial screening approach, although the molecular mechanisms remain to be elucidated. The combination of ibrutinib with either proteasome or BCL-2 inhibition is proposed as promising novel treatments for MCL and CLL. A phase I/II clinical trial has been designed to test ibrutinib + carfilzomib in relapsed or refractory MCL and CLL. Disclosures:Williams:Celgene: Consultancy, Independent Data Safety Committee member Other, Research Funding; Genentech: Consultancy, Research Funding; Janssen: Research Funding; Millennium: Consultancy, Independent Data Safety Committee member, Independent Data Safety Committee member Other, Research Funding; Pharmacyclics: Consultancy, Research Funding; Onyx: Research Funding; Gilead: Research Funding. Wang:Onyx: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Millennium: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Novartis: Research Funding.

Pharmacologic Targeting of Mcl-1 Induces Mitochondrial Dysfunction and Apoptosis in B-Cell Lymphoma Cells in a TP53- and BAX-Dependent Manner.

Bcl-2 has been effectively targeted in lymphoid malignancies. However, resistance is inevitable, and novel approaches to target mitochondrial apoptosis are necessary. AZD5991, a selective BH3-mimetic in clinical trials, inhibits Mcl-1 with high potency. We explored the preclinical activity of AZD5991 in diffuse large B-cell lymphoma (DLBCL) and ibrutinib-resistant mantle cell lymphoma (MCL) cell lines, MCL patient samples, and mice bearing DLBCL and MCL xenografts using flow cytometry, immunoblotting, and Seahorse respirometry assay. Cas9 gene editing and ex vivo functional drug screen assays helped identify mechanisms of resistance to Mcl-1 inhibition. Mcl-1 was expressed in DLBCL and MCL cell lines and primary tumors. Treatment with AZD5991 restricted growth of DLBCL cells independent of cell of origin and overcame ibrutinib resistance in MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction as manifested by mitochondrial membrane depolarization, decreased mitochondrial mass, and induction of mitophagy. This was accompanied by impairment of oxidative phosphorylation. TP53 and BAX were essential for sensitivity to Mcl-1, and oxidative phosphorylation was implicated in resistance to Mcl-1 inhibition. Induction of prosurvival proteins (e.g., Bcl-xL) in stromal conditions that mimic the tumor microenvironment rendered protection of primary MCL cells from Mcl-1 inhibition, while BH3-mimetics targeting Bcl-2/xL sensitized lymphoid cells to AZD5991. Treatment with AZD5991 reduced tumor growth in murine lymphoma models and prolonged survival of MCL PDX mice. Selective targeting Mcl-1 is a promising therapeutic approach in lymphoid malignancies. TP53 apoptotic network and metabolic reprogramming underlie susceptibility to Mcl-1 inhibition.