Primary Mantle Cell Lymphoma Research Articles

Extranodal Mantle Cell Lymphoma Presented as Bilateral Adrenal Masses on 18F-FDG PET/CT and 68Ga-DOTATATE PET/CT.

Mantle cell lymphoma (MCL) is a rare type of B-cell non-Hodgkin lymphoma that commonly presents with constitutional symptoms, lymphadenopathy, and laboratory abnormalities. Although less common, extranodal involvement of MCL may be seen at initial presentation and most commonly occurs in the spleen, liver, bone marrow, and gastrointestinal tract. Adrenal involvement in MCL is extremely rare, and only a few cases have been reported in the literature. Herein, we present 18F-FDG PET/CT and 68Ga-DOTATATE PET/CT findings of a unique case of primary extranodal MCL with gastric and adrenal involvement.

Targeting CERS6-AS1/FGFR1 axis as synthetic vulnerability to constrain stromal cells supported proliferation in Mantle cell lymphoma.

The interaction between stromal and tumor cells in tumor microenvironment is a crucial factor in Mantle cell lymphoma (MCL) progression and therapy resistance. We have identified a long non-coding RNA, CERS6-AS1, upregulated in MCL and associated with poor overall survival. CERS6-AS1 expression was elevated in primary MCL within stromal microenvironment and in a subset of MCL cells adhered to stromal layer. These stromal-adhered MCL-subsets exhibited cancer stem cell signatures than suspension counterparts. Mechanistically, we found that downregulating CERS6-AS1 in MCL reduced Fibroblast Growth Factor Receptor-1 (FGFR1), expression attributed to loss of its interaction with RNA-binding protein nucleolin. In addition, using in-silico approach, we have discovered a direct interaction between nucleolin and 5'UTR of FGFR1, thereby regulating FGFR1 transcript stability. We discovered a positive association of CERS6-AS1 with cancer stem cell signatures, and Wnt signaling. Building on these, we explored potential therapeutic strategies where combining nucleolin-targeting agent with FGFR1 inhibition significantly contributed to reversing cancer stem cell signatures and abrogated primary MCL cell growth on stromal layer. These findings provide mechanistic insights into regulatory network involving CERS6-AS1, nucleolin, and FGFR1 axis-associated crosstalk between tumor cells and stromal cell interaction and highlights therapeutic potential of targeting a non-coding RNA in MCL.

Abstract 602: Targeting CDK9 with a novel potent and selective inhibitor, YX0798, in CAR-T-relapsed mantle cell lymphoma models

Abstract Introduction: Cyclin-dependent kinase (CDK) family members play critical roles in multiple cellular processes. CDK9 is a central regulator at several stages of PolII transcription, including initiation, elongation, and termination. CDK9 dysfunction resulted in transcriptomic reprogramming and promotion of the development of cancers, including lymphoma and leukemia. We and others reported that transcriptomic reprogramming is associated with disease progression and therapeutic resistance in mantle cell lymphoma (MCL). Targeting CDK9 with its inhibitors AZD4573 and enitociclib is safe and effective for treatment in preclinical MCL models, and they are currently under clinical investigation to assess their efficacy and safety. The data demonstrated that CDK9 is a promising therapeutic target. In this study, we developed a novel CDK9 inhibitor, YX0798, and assessed its potency and safety in preclinical MCL models. Method: Cell viability assays were conducted in a time- and dose-dependent manner to assess the in vitro efficacy of YX0798 as well as AZD4573. Apoptosis assays and western blots were used to determine the mechanism of action in MCL cells. Patient-derived xenograft models from primary MCL patient samples were established in immunodeficient mice and used to determine the therapeutic potential YX0798. Results: Next generation sequencing showed that CDK9 was upregulated and cancer hallmarks MYC-TARGETS-v1 and -v2 were highly enriched in CAR-T-relapsed MCL patients compared to CAR-T-naïve patients. We hypothesized that CDK9 overexpression and activity may greatly contribute to the enrichment of MYC-TARGETS-v1 and -v2 in patients with CAR-T resistance, so targeting CDK9 may overcome that resistance. YX0798 was developed to be a potent and selective CDK9 inhibitor. It markedly inhibited cell viability and induced robust apoptosis in MCL cell lines. As expected, it inhibited CDK9 kinase activity and suppressed the expression of short-lived proteins like c-MYC and MCL-1 that are critical for MCL cell survival and growth. YX0798 at 2 mg/kg for continuous 7 days via pump or at 5 mg/kg daily by oral administration potently inhibited the tumor growth of xenografts derived from a patient with therapeutic relapse to the CAR-T therapy in vivo (P<0.0001). This treatment efficacy was found to be similar at that of AZD4573 but at a higher dosage (15+15 mg/kg, 2 hours split, QW, IP). No adverse effects in mice were observed for any of treatments. Conclusion: These data show that our novel CDK9 inhibitor YX0798 is potent and efficacious in treating aggressive MCL models and overcame CAR-T resistance at a lower dosage than AZD4573. Citation Format: Vivian Jiang, Yu Xue, Hong Kim, Joseph McIntosh, Haiying Chen, Tianci Zhang, Yang Liu, Jia Zhou, Michael Wang. Targeting CDK9 with a novel potent and selective inhibitor, YX0798, in CAR-T-relapsed mantle cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 602.

B-cell receptor signaling activity identifies patients with mantle cell lymphoma at higher risk of progression

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

The dual HCK/BTK inhibitor KIN-8194 impairs growth and integrin-mediated adhesion of BTKi-resistant mantle cell lymphoma

Although Bruton’s tyrosine kinase (BTK) inhibitors (BTKi) have significantly improved patient prognosis, mantle cell lymphoma (MCL) is still considered incurable due to primary and acquired resistance. We have recently shown that aberrant expression of the Src-family tyrosine kinase hematopoietic cell kinase (HCK) in MCL correlates with poor prognosis, and that genetic HCK perturbation impairs growth and integrin-mediated adhesion of MCL cells. Here, we show that KIN-8194, a dual inhibitor of BTK and HCK with in vivo activity against Myd88-L265P-driven diffuse large B-cell lymphoma and Waldenström Macroglobulinemia, has a potent growth inhibitory effect in MCL cell lines and primary MCL cells, irrespective of their sensitivity to BTKi (ibrutinib and acalabrutinib). In BTKi-resistant cells this is mediated by inhibition of HCK, which results in repression of AKT-S6 signaling. In addition, KIN-8194 inhibits integrin-mediated adhesion of BTKi-sensitive and insensitive MCL cells to fibronectin and stromal cells in an HCK-dependent manner. Finally, we show that MCL cells with acquired BTKi resistance retain their sensitivity to KIN-8194. Taken together, our data demonstrate that KIN-8194 inhibits growth and integrin-mediated adhesion of BTKi-sensitive MCL cells, as well as MCL cells with primary or acquired BTKi resistance. This renders KIN-8194 a promising novel treatment for MCL patients.

Exploring BiTE-Integrated CAR T-Cell Therapy to Overcome Tumor Antigen Escape and Reinforce CAR-T Therapy in Mantle Cell Lymphoma

Background T cell engagers (BiTEs) are bispecific T-cell engaging antibodies that redirect T cells to target antigen-expressing tumor cells. Combining a second antigen-targeted bispecific T cell engager (BiTE) with CAR T cells or incorporating bispecific engaging antibody-secreting T cells (engager-T cells) is expected to augment T-cell activation and proliferative capacity, and antitumor cytotoxicity, even towards tumor cells expressing tumor antigens heterogeneously. Here, we report a BiTE-integrated T-cell therapeutic strategy to overcome tumor antigen escape and reinforce the efficacy of CAR-T therapy in mantle cell lymphoma (MCL). Methods CD20-directed bispecific T-cell engager (CD20-BiTE) was constructed with a publicly available VHH fragment from an anti-CD20 single-domain antibody, and anti-CD3 scFv from OKT3 monoclonal antibody. CD19-directed CAR construct is comprised of anti-CD19 scFv from publicly available FMC63 monoclonal antibody, CD8 transmembrane-, 4-1BB costimulatory-, and CD3ζ signaling-domains. Human primary pan-T cells were isolated from PBMC derived from normal human donors (MD Anderson Blood Bank) or patients with MCL. Activated T cells were used for effector functional assay or transduced with lentivirus to produce CAR T or engager-T cells. Effector function of bispecific antibodies and T cells was determined via T-cell activation, cytokine release, and T-cell-dependent cytotoxicity assay in a co-culture model. Results Treatment with CD20-BiTE enhances the effector function of naïve T cells: the BiTE-Fc fusion protein was ectopically expressed in Expi293 and purified by immobilized Protein-A. The binding specificity of scFv(s) of CD20-BiTE was confirmed to bind strongly and specifically to CD20-expressing cells. In vitro functional analyses demonstrated that co-culture of naïve T cells with CD20-exressing cells in the presence of CD20-BiTE induced antigen-specific activation of the T cells (increase in the expression of CD25 and CD69). In the presence of naïve T cells and antigen-positive MCL cells, CD20-BiTE enhanced the secretion of cytokines (IFNγ and TNFα, > 2-fold increase) and CD8 T-cell cytotoxic molecule granzyme B by T cells, in a BiTE-dependent and CD20-specific manner. The enhanced effector function translated into improved antitumor cytotoxicity, as activated T cells treated with CD20-BiTE exerted 43- 72% increase in the killing efficacy of JeKo-1 BTK-KD cells. CD20-BiTE-combined CAR T cells endowed with enhanced antitumor activity overcome tumor antigen escape and resistance to current CD19-CAR T cell therapy: when primary T cells and CD19-CAR T cells were co-cultured with MCL cells, addition of CD20-BiTE potentiated T cell-dependent cytotoxicity ( p < 0.001). Since both CD19-dependent and -independent CAR T cell-resistance often develop during adoptive cell therapy in MCL, we sought to overcome the resistance in MCL by combining a second antigen-targeted CD20-BiTE with CAR T cells or incorporating CD20-BiTE secreting T cells (engager-T cells). Remarkably, when combined with CD19-CAR T cells, CD20-BiTE mediates significant increase in the antitumor cytotoxicity, compared to T cells alone ( p ≤ 0,001 and p < 0.001 for 2nM and 5nM of the BiTE, respectively), in a CD20-specific fashion, in both MCL cells with diminished CD19 expression, and CD19-CAR T-resistant primary MCL cells. Importantly, in a transwell assay, CD19-CAR T cells demonstrated restored potent killing (>3-fold increase over CAR T cells alone) of the CD19-low or CAR T therapy resistant tumor cells, but only when the engager-T cells were present in the insert, suggesting the engaging molecules were able to redirect CD19-CAR T cells to CD20-positive tumor cells regardless of their CD19 expression. The anti-lymphoma efficacy of engager-CAR T cell therapy will be further evaluated in our well-established unique PDX model for MCL. In summary, we have successfully generated a CD20-directed bispecific T-cellengaging antibody and CD20-BiTE engager-T cells that could be combined with CAR T cell therapy, which was demonstrated effective in overcoming tumor antigen escape and resistance to current CD19-CAR T cell therapy in MCL.

A collision tumor of nasopharyngeal carcinoma and primary mantle cell lymphoma in the nasopharynx: a case report and review of the literature

BackgroundNasopharyngeal carcinoma (NPC) is more common in men aged 40 to 59, and radiotherapy is an effective treatment. Nasopharyngeal lymphoma (NPL) is rare, and the coexistence of nasopharyngeal mantle cell lymphoma (MCL) and NPC is even rarer. A collision tumor is a rare type of tumor that refers to two or more different tumors occurring in the same organ. No reports to date have described a collision tumor of NPC and MCL occurring within the same nasopharyngeal mass. We herein report the successful treatment of a unique case of synchronous coexistence of NPC and MCL occurring in the nasopharynx of a Chinese man.Case presentationA 58-year-old man presented with a 5-month history of swallowing discomfort. Biopsy was performed under nasopharyngeal endoscopy, and histopathology revealed NPC. Magnetic resonance imaging revealed lesions in the nasopharynx, oropharynx, and tonsils, as well as enlarged lymph nodes in the parotid gland, posterior ear, and neck. This may be a synchronous dual primary tumor coexisting with NPC and NPL. Pathology consultation confirmed that the biopsy specimen of the nasopharynx was a collision tumor of NPC and MCL. Positron emission tomography computed tomography (PET-CT) revealed thickening of the posterior wall of the nasopharynx, which was considered NPC with lymphoma. The enlargement of the pharyngeal lymph ring and multiple hypermetabolic lymph nodes were evaluated as lymphoma infiltration. The patient received two courses of R-CHOP chemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) followed by head and neck radiotherapy. At the time of this writing, he had remained alive without recurrence for 61 months since the initial treatment and was still undergoing follow-up.ConclusionsIt is very important to correctly recognize collision tumors. Magnetic resonance imaging helps identify different components of collision tumors. Pathological examination helps to confirm the diagnosis. Histological examination reveals different components, and PET-CT can help determine the extent of the lesion. Dose-adjusted chemotherapy combined with radiotherapy may have promising herapeutic effects, but additional case studies are needed to confirm.

Primary Mantle Cell Lymphoma of Rectum with Rare Presentation: A Unique Case Report and Review of Literature

Primary Mantle Cell Lymphoma of Rectum with Rare Presentation: A Unique Case Report and Review of Literature

Combined treatment with crizotinib and temsirolimus is an effective strategy in mantle cell lymphoma and can overcome acquired resistance to temsirolimus.

Constitutive activation of the PI3K/AKT/mTOR-pathway plays an important role in the pathogenesis of mantle cell lymphoma (MCL), leading to approval of the mTOR inhibitor temsirolimus for relapsed or refractory MCL. Yet, despite favorable initial response rates, early relapses under treatment have been observed. Therefore, understanding the underlying mechanisms of temsirolimus resistance and developing strategies to overcome it is highly warranted. Here, we established a new temsirolimus-resistant MCL cell line to evaluate the molecular background of resistance to this drug. Transcriptome profiling and gene set enrichment analysis comparing temsirolimus-sensitive and -resistant cell lines showed significant upregulation of PI3K/AKT/mTor-, RAS signaling- and the RTK-dependent PDGFR-, FGFR-, Met- and ALK-signaling-pathways in the resistant cells. Furthermore, MET, known as important proto-oncogene and mediator of drug resistance, was among the most upregulated genes in the resistant cells. Importantly, Met protein was overexpressed in both, MCL cells with acquired as well as intrinsic temsirolimus resistance, but could not be detected in any of the temsirolimus sensitive ones. Combined pharmacological inhibition of mTOR and Met signaling with temsirolimus and the RTK inhibitor crizotinib significantly restored sensitivity to temsirolimus. Furthermore, this combined treatment proved to be synergistic in all MCL cell lines investigated and was also active in primary MCL cells. In summary, we showed for the first time that overexpression of MET plays an important role for mediating temsirolimus resistance in MCL and combined treatment with temsirolimus and crizotinib is a very promising therapeutic approach for MCL and an effective strategy to overcome temsirolimus resistance.

MYC-induced cytidine metabolism regulates survival and drug resistance via cGas-STING pathway in mantle cell lymphoma.

Lymphocyte proliferation and tumourigenesis are dependent on nucleotide synthesis to support DNA, RNA and phospholipid synthesis. Here, we identified that reprogramming of nucleotide metabolism as an important factor divides mantle cell lymphoma (MCL) into two groups with different transcriptional signalling pathways and varying prognoses. We establish a nucleotide metabolism-related prognostic model that includes six genes with different regression coefficients, which significantly predicts prognosis for MCL patients (p < 0.0001). Of these six genes, de novo CTP synthesis pathway enzyme CTPS1 whose inhibitor (STP938) is already in clinical trials for relapsed/refractory lymphomas (NCT05463263) has the highest regression coefficient. An increase in CTPS1 expression predicts adverse overall survival and progression-free survival with independent prognostic significance in 105 primary MCL samples and GEO database (GSE93291). CRISPR CTPS1 knockout causes DNA damage and proliferation defects in MCL. Additionally, MYC positively regulates CTPS1 expression, and TP53-aberrant and ibrutinib-resistant MCL cells also rely on cytidine metabolism. Furthermore, besides the obvious decreased CTP pool caused by CTPS1 deficiency, CTPS1 inhibition may also induce immune-related responses via activating dsDNA-cGAS-STING pathway, which plays a crucial role in impeding tumour growth in MCL patients.

Roles of PI3Kγ and PI3Kδ in mantle cell lymphoma proliferation and migration contributing to efficacy of the PI3Kγ/δ inhibitor duvelisib

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma that is incurable with existing therapies, and therefore presents a significant unmet clinical need. The ability of this disease to overcome therapy, including those that target the B cell receptor pathway which has a pathogenic role in MCL, highlights the need to develop new treatment strategies. Herein, we demonstrate that a distinguishing feature of lymph node resident MCL cells is the expression of phosphatidylinositol 3-kinase γ (PI3Kγ), a PI3K isoform that is not highly expressed in other B cells or B-cell malignancies. By exploring the role of PI3K in MCL using different PI3K isoform inhibitors, we provide evidence that duvelisib, a dual PI3Kδ/γ inhibitor, has a greater effect than PI3Kδ- and PI3Kγ-selective inhibitors in blocking the proliferation of primary MCL cells and MCL cell lines, and in inhibiting tumour growth in a mouse xenograft model. In addition, we demonstrated that PI3Kδ/γ signalling is critical for migration of primary MCL cells and cell lines. Our data indicates that aberrant expression of PI3Kγ is a critical feature of MCL pathogenesis. Thus, we suggest that the dual PI3Kδ/γ duvelisib would be effective for the treatment of mantle cell lymphoma.

CD52 and OXPHOS—potential targets in ibrutinib-treated mantle cell lymphoma

Altered features of tumor cells acquired across therapy can result in the survival of treatment-resistant clones that may cause minimal residual disease (MRD). Despite the efficacy of ibrutinib in treating relapsed/refractory mantle cell lymphoma, the obstacle of residual cells contributes to relapses of this mature B-cell neoplasm, and the disease remains incurable. RNA-seq analysis of an ibrutinib-sensitive mantle cell lymphoma cell line following ibrutinib incubation of up to 4 d, corroborated our previously postulated resistance mechanism of a metabolic switch to reliance on oxidative phosphorylation (OXPHOS) in surviving cells. Besides, we had shown that treatment-persisting cells were characterized by increased CD52 expression. Therefore, we hypothesized that combining ibrutinib with another agent targeting these potential escape mechanisms could minimize the risk of survival of ibrutinib-resistant cells. Concomitant use of ibrutinib with OXPHOS-inhibitor IACS-010759 increased toxicity compared to ibrutinib alone. Targeting CD52 was even more efficient, as addition of CD52 mAb in combination with human serum following ibrutinib pretreatment led to rapid complement-dependent-cytotoxicity in an ibrutinib-sensitive cell line. In primary mantle cell lymphoma cells, a higher toxic effect with CD52 mAb was obtained, when cells were pretreated with ibrutinib, but only in an ibrutinib-sensitive cohort. Given the challenge of treating multi-resistant mantle cell lymphoma patients, this work highlights the potential use of anti-CD52 therapy as consolidation after ibrutinib treatment in patients who responded to the BTK inhibitor to achieve MRD negativity and prolong progression-free survival.

Tumorigenic role of Musashi-2 in aggressive mantle cell lymphoma

SOX11 overexpression has been associated with aggressive behavior of mantle cell lymphomas (MCL). SOX11 is overexpressed in embryonic and cancer stem cells (CSC) of some tumors. Although CSC have been isolated from primary MCL, their relationship to SOX11 expression and contribution to MCL pathogenesis and clinical evolution remain unknown. Here, we observed enrichment in leukemic and hematopoietic stem cells gene signatures in SOX11+ compared to SOX11– MCL primary cases. Musashi-2 (MSI2) emerged as one of the most significant upregulated stem cell-related genes in SOX11+ MCLs. SOX11 is directly bound to the MSI2 promoter upregulating its expression in vitro. MSI2 intronic enhancers were strongly activated in SOX11+ MCL cell lines and primary cases. MSI2 upregulation was significantly associated with poor overall survival independently of other high-risk features of MCL. MSI2 knockdown decreased the expression of genes related to apoptosis and stem cell features and significantly reduced clonogenic growth, tumor cell survival and chemoresistance in MCL cells. MSI2-knockdown cells had reduced tumorigenic engraftment into mice bone marrow and spleen compared to control cells in xenotransplanted mouse models. Our results suggest that MSI2 might play a key role in sustaining stemness and tumor cell survival, representing a possible novel target for therapeutic interventions in MCL.

DNMT3A-Mediated Oxidative Phosphorylation and Ibrutinib Resistance in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL), a B cell non-Hodgkin lymphoma, is incurable with current treatment modalities. Targeting Bruton tyrosine kinase (BTK) in the early B cell receptor signaling pathway by ibrutinib has shown remarkable anti-tumor activity, with the overall response rate of 68% in relapsed/refractory MCL patients. However, almost all the responders relapsed after ibrutinib treatment, with the 1-year survival rate of 22%. To overcome primary and acquired resistance to ibrutinib, novel treatment strategies are urgently needed. The presence of a relapse-specific C481S mutation in BTK associated with acquired ibrutinib resistance has been frequently identified in chronic lymphocytic leukemia but is rare in clinical MCL specimens based on sequencing results. Thissuggests that non-genetic aberrations likely play a role in ibrutinib resistance. The recent emergence of mitochondrial oxidative phosphorylation (OXPHOS) as a driver of ibrutinib resistance warrants the necessity to identify the components that govern OXPHOS in MCL. This study identifies the de novo DNA methyltransferase 3A (DNMT3A) as a mediator of mitochondrial biogenesis and OXPHOS in MCL. Using multiomics and functional approaches, we found that DNMT3A is required and sufficient to confers ibrutinib resistance in MCL. We show that DNMT3A promotes MCL growth by enhancing mitochondrial biogenesis, mitochondrial functions and OXPHOS. These effects are mediated by its interaction with MEF2B, a transcriptional activator of MYC, to enhance MYC-mediated transcription of genes involved in mitochondrial biogenesis. More importantly, the level of DNMT3A protein increases after ibrutinib treatment in primary MCL, which is consistent with a previous finding that the expression of DNMT3A was higher in ibrutinib-resistant than in ibrutinib-sensitive MCL patients. We demonstrate that DNMT3A knockout sensitizes ibrutinib-resistant cells to ibrutinib treatment. Conversely, the overexpression of DNMT3A cDNA de-sensitizes ibrutinib-sensitive cell lines. Interestingly, the overexpression of a catalytically dead DNMT3A confers resistance to ibrutinib in ibrutinib-sensitive cell lines, suggesting that DNMT3A-mediated ibrutinib resistance is independent of its enzymatic activity. Finally, we demonstrate that targeting DNMT3A by a low dose of decitabine, which induces the degradation of DNMT3A protein, synergizes with IM156, an inhibitor of mitochondrial complex I and currently in phase I clinical trial, in killing ibrutinib-resistant cell lines and primary MCL cells. Currently, we are testing this synergistic effect using patient-derived xenograft mouse models. Therefore, our study reveals a novel noncatalytic function of DNMT3A and suggests that DNMT3A is a potential biomarker and molecular target in treating MCL (Figure 1). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Sumoylation with Subasumstat (TAK-981), a Selective Inhibitor of SUMO-Activating Enzyme (SAE), Induces Mitochondrial Dysfunction in Preclinical Models of Non-Hodgkin Lymphoma (NHL)

SUMOylation is a post-translational modification which leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to lysine residues of a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. Subasumstat, (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 B-cell non-Hodgkin's lymphoma (NHL). TAK-981 was provided by Takeda Development Center Americas, Inc. (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines (OCI-LY3, VAL, U-2932, SU-DHL10), 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 cell lines with an IC50~10 nM. SAEi led to rapid (within 2 h) protein desumoylation in a dose dependent manner, prominently in the ~90 kDa region. TAK-981 induced pronounced double strand DNA breaks in DLBCL cells (comet assay) and cell cycle arrest (propidium iodine). RNA-Seq and ATAC-Seq analysis of TAK-981-treated DLBCL cells revealed upregulation of the DNA damage, NF-κB, and OXPHOS signaling pathways. We noted that in addition to cytoplasmic proteins, 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 (by electron microscopy) and a change of mitochondrial dynamics as assessed by expression of mitochondrial proteins (eg, Drp1). Seahorse respirometry analysis revealed dramatic downmodulation of OXPHOS following SAEi. Furthermore, metabolomic profiling of DLBCL cells treated with TAK-981 showed downmodulation of TCA substrates, while glycolysis substrates remained unchanged. The mitochondrial disruption observed following TAK-981 treatment was accompanied by rapid accumulation of reactive oxygen species, a mechanism that has been previously shown to lead to DNA damage in cancer cells. Consistent with cell line data, 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, DNA damage and centromere/telomere gene 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, we used DLBCL xenografts. OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received 10 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 analysis. Conclusions. 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.

Combined Inhibition of CDK2 and BCL2 Overrides Resistance to Targeting BTK and CDK4/6 in Mantle Cell Lymphoma Therapy

Mantle cell lymphoma (MCL) remains incurable due to the development of drug resistance, despite the plethora of therapies available. Each successive treatment failure is associated with a more rapidly proliferating disease and fewer treatment options. Understanding the drug-resistance mechanism and developing effective, well-tolerated, new therapies for MCL are urgently needed. Unrestrained proliferation of MCL cells is mainly driven by aberrant cyclin D1 expression and dysregulated CDK4 activity that promotes cell cycle progression from G1 to S. In our phase 1 clinical trial inhibiting CDK4/6 with palbociclib and BTK with ibrutinib (PALIBR) in recurrent MCL, the complete response rate (CR) was 42% compared to 21% in response to ibrutinib alone, despite a comparable 67% overall response rate. Moreover, 5 patients remain on therapy (2 CR and 3 PR) for ~ 8 years, suggesting that CDK4/6 inhibition deepens and prolongs the clinical response to BTK inhibition in MCL. However, resistance emerges in some patients. To elucidate the underpinnings, we undertook integrative longitudinal genomic analysis of sequential tissue and blood specimens (n=53) of 27 MCL patients before, during therapy and on progression, and 4 treatment-naïve MCL patients and 4 normal subjects as controls. Whole exome and whole transcriptome sequencing of freshly isolated MCL cells and B cells revealed that resistance to PALIBR is rooted in homozygous deletion of RB1 (direct target of CDK4/6) or compound copy number variation (cCNV) - concurrent hemizygous loss of RB1 and CDKN2A (p16, CDK4/6 inhibitor) and gain of CDK4. In other resistant patients, the RB protein is silenced by translational termination mutation. Single-cell RNA-sequencing (scRNA-seq, 210,000 cells) of corresponding PBMCs using a unique in-house MCL RNA reference library reveals that MCL cells comprise 4 transcriptomically distinct clusters. Cluster 1 (C1) is similar to quiescent normal B cells; C2 resembles hyper-activated B cells enriched for signatures of BCR and cytokine signaling; C3 represents non-proliferating, long-lived MCL cells; and C4 is highly proliferative, expanding with disease progression. RNA velocity analysis further uncovered that C2 fuels the transition to C4 with disease progression, whereas C3 is a terminal node (Fig 1). In C2 and C3 MCL cells, CDK2 is highly elevated, indicating evasion of CDK4/6 inhibition, and PIK3IP1 (negative PI3K regulator) is silenced, suggesting that compensatory PI3K activation in BTKi resistance (Chiron et al, Cancer Discovery 2014) is secondary to loss of cell cycle control. BCL2 is markedly elevated in C2, greater in C3 MCL cells along with increases in anti-apoptotic BCL2L1(BCL-XL) and BCL-2A1 and decreases in pro-apoptotic PMAIP1 (NOXA) expression. This suggests combined CDK2 and BCL2 inhibition as a rational approach to override CDK4/6i and BTKi resistance. Indeed, combined inhibition of CDK2 with PF-07104091 and BCL2 with venetoclax (PF-VEN) cooperatively impaired growth and killed ibrutinib-resistant MAVER-1 cells, more effectively in the isogenic derivative MAVER-1R cells which we generated to recapitulate cCNV in PALIBR resistance - depletion of RB protein through translational termination mutation (W99, AAF 75%) in RB1, loss of CDKN2A and gain of CDK4. Mechanistically, MAVER-1R cells are accelerated in G1 to S progression, based on elevation of CDK2 and TK1 expression and cell cycle profiling. They are intrinsically apoptotic and vulnerable to BCL2 inhibition due to a marked reduction in cells expressing anti-apoptotic BCL2L1 and IRF4, and universal expression of pro-apoptotic PMAIP1 (NOXA). Importantly, combined CDK2 and BCL2 inhibition synergistically kills MCL cells of a patient resistant to PALIBR ex vivo. In summary, by longitudinal genomic analysis, we have provided the first evidence that 1) resistance to CDK4/6i and BTKi stems from MCL intrinsic cCNV; 2) MCL cells comprise 4 major transcriptomically distinct clusters; cluster 2 expressing copious CDK2 and BCL2 is pivotal in fueling proliferation of MCL cells in resistance; 3) cluster 3 cells also accumulate with resistance due to longevity; 4) combined inhibition of CDK2 and BCL2 overrides resistance to CDK4i and BTKi, in MCL cell lines and primary MCL cells from PALIBR resistant patient. Combined inhibition of CDK2 and BCL2, therefore, represents a mechanism-based strategy to overcome CDK4/6i and BTKi resistance in MCL therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

STP938, a Selective CTPS1 Inhibitor, Shows Single Agent Activity and Synergy with BCL2 Inhibition in Preclinical Models of Mantle Cell Lymphoma

Background Proliferating tumor cells are dependent on nucleotide synthesis to support DNA replication and RNA synthesis. The homologous CTP synthetases, CTPS1 and CTPS2, play a central role by catalyzing the rate-limiting conversion of UTP to CTP. Interestingly, while most tissues rely on CTPS2, lymphoid cells depend on CTPS1, as observed in rare patients with inherited CTPS1 loss of function manifesting selective B/T cell defects but no non-hematopoietic phenotype (Martin 2014, PMID 24870241). Herein, we investigate inhibition of CTPS1, alone or in combination, as a potential novel therapeutic target in mantle cell lymphoma (MCL). Methods STP938 (Step Pharma) is a highly selective small molecule inhibitor of CTPS1 with >1,300-fold selectivity over CTPS2. In vitro cell proliferation was assessed at 72 hours by CellTiter-Glo. Synergy was quantified by Bliss scores. 3'seq-RNA profiling was performed before and after a 24-hour exposure to STP938; normalized data were subjected to KEGG pathway enrichment analysis. In vivo efficacy was established following subcutaneous transplantation of MCL cells (Z138) into immunodeficient mice (NSG, n=5 per treatment group); treatment was initiated when tumors reached approximately 50 mm3. STP938 was dosed at 30 mg/kg/day subcutaneously days 1-4 of a 7 day cycle; venetoclax at 75 mg/kg/day orally days 2-5 of a 7 day cycle for three cycles. Tumor size was assessed by calipers. Results Analysis of the Achilles CRISPR dataset identified a dependency of human B cell lymphoma cell lines (n=34) on the de novo but not the salvage pyrimidine synthesis pathway. Of note, whilst CRISPR deletion of CTPS1 resulted in a marked proliferation defect, CTPS2 loss had no effect, consistent with the central role for CTPS1 in lymphoid cells established in human genetic studies. Analysis of publicly available data from primary MCL samples (n=122, GSE93291) identified high CTPS1 expression as a predictor of inferior overall survival (P=<0.0001). A selective CTPS1 inhibitor, STP938, was used to evaluate a potential therapeutic role for CTPS1 inhibition. Of 15 MCL cell lines tested, 6 (40%) were highly sensitive to STP938 (IC50 <0.1 μM), 5 (33%) showed intermediate sensitivity (IC50 0.1 - 1 μM) and 4 (27%) were resistant (IC50 >1 μM). Of note, sensitivity to STP938 was independent of known indicators of poor prognosis, such as TP53 mutation, and was independent of resistance to standard of care therapies such as ibrutinib or venetoclax. Pathway analysis of transcriptomic data (n=9 cell lines) identified downregulation of cell cycle pathways in response to STP938 exposure. Directed analysis identified modulation of BCL2-family transcripts, with STP938 inducing increases in BCL2 and BCL2L1 (BCL-XL) expression and a decrease in MCL1 expression (P=0.02, 0.006 and 0.02, respectively; paired t-test). BCL2 increase and MCL1 decrease upon CTPS1 inhibition have been further confirmed at the protein level (24h, 20nM). Given these findings, in vitro combination studies were undertaken with a selective BCL2 inhibitor. STP938 plus venetoclax demonstrated synergy in 5 of 9 MCL cell lines tested (Figure A). Of note, there was no association between synergy and sensitivity to single agent STP938, with two cell lines resistant to single agent STP938 (IC50 3.1 and >10 μM) nonetheless showing synergy with venetoclax. The synergy between STP938 and venetoclax was further investigated in an in vivo model of MCL. Combination therapy was well tolerated. Single agent venetoclax showed no significant inhibition of tumor growth. Single agent STP938 slowed tumor growth to approximately 50% of vehicle control. The combination of STP938 and venetoclax showed strong synergy with more than 80% inhibition of tumor growth for 3 cycles of treatment (Figure B). Conclusions Dependence of tumor cells on elevated rates of nucleotide synthesis has been exploited by agents such as cytarabine. Whilst therapy for hematological cancers is moving away from cytotoxic drugs in favor of targeted agents, inhibition of nucleotide synthesis remains an attractive target. STP938, a highly selective CTPS1 inhibitor, demonstrates single agent activity in vitro and in vivo, and shows strong synergy with the approved BCL2 inhibitor venetoclax. STP938 will enter clinical development for relapsed refractory B and T cell lymphomas in 2022. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CARD11 and BCL2A1 Join Forces to Promote Resistance to Ibrutinib/Venetoclax Combination in Lymphoma Patients

Background Mantle cell lymphoma (MCL) is a hard to cure malignancy with conventional chemotherapy. We previously reported that combinations targeting CD20 (obinutuzumab), BTK (ibrutinib) and BCL2 (venetoclax) counteracted both tumor intrinsic anomalies and dialogs within corrupted MCL ecosystems. Importantly, this chemo-free combination provided durable complete responses in patients both at relapse or naïve of treatments (OAsIs trial, NCT02558816). Despite high initial response rates, nearly 1/3 of patients rapidly relapsed. To understand this in vivo resistance, we performed targeted sequencing and single-cell RNA-seq to identify key actors involved. Methods Tumor DNA from patients enrolled in the OAsIs trial were sequenced at baseline (n=12) or relapse (n=5) (Capture xGen technology, IDT). Forty-nine genes and the 9p21 region, described for their impact in lymphoma resistance, were targeted for recurrent SNVs/CNVs. Single cell RNA-seq (3' gene expression, 10X genomics) was performed before treatment (n=4) or after relapse (n=3). Integrated omics analysis was performed to define resistance signatures, at the single cell resolution and associated to gain of function (GOF) mutations. Functional analysis (i.e., reporter assay, BH3-profiling, drug testing) using primary MCL cells and cell lines were performed. Results Targeted sequencing of tumor cells at relapse highlighted an enrichment of CARD11 GOF mutations (p=0.01). The selective advantage of CARD11MUT cells under OAsIs pressure was further documented with a patient who achieved a complete response before relapsing 12 months after inclusion. Whereas CARD11MUT tumor cells were in a minority (0.0005 %) at the onset of treatment, all the cells carried a heterozygous mutation at relapse (VAF: 50.56%). scRNA-seq of the same longitudinal samples identified a minor cluster (187/4860 sequenced cells) enriched at relapse that was characterized by expression of NFKB1 target genes consistent with constitutive CARD11 activity (Enrichr p<5x10-4). By combining scRNA-seq and mutations, we identified a signature associated with CARD11 GOF, which we named "OAsIs-R" signature. Applied to additional patients' samples, this novel signature allowed the segregation of OAsIs sensitive from resistant cells (scRNA-seq). Of major interest, OAsIs-R signature was also predictive for OS/PFS in MCL patients treated with conventional chemotherapy (GSE93291 and in-house cohorts). Among the top genes of OAsIs-R signature, we further focused on BCL2A1. We first highlighted its frequent expression in MCL primary cells (n=63) and cell lines (n=7). BCL2A1 overexpression, BH3-profiling (FS2 peptide) and ex vivo drug testing (AnnexinV) further validated a key role of CARD11-related BCL2A1 dysregulation in OAsIs resistance. Ibrutinib deeply reduced BCL2A1 level in CARD11WT cell lines but not in CARD11MUT cells (RNA expression inhibition: 82%, n=4 vs. 0%, n=4), leading to synergistic apoptosis when associated to venetoclax in BCL2A1+ but not in BCL2A1- cells (mean synergy score: 17 vs. 1, n=6). Using reporter assay experiments we showed that CARD11 mutations directly led to an increase activity of BCL2A1 promoter (2 fold more activity, Mut vs. wt), reinforcing the need to target its activity to counteract resistance. Among druggable strategies to inhibit CARD11, selective inhibition of its partner MALT1, essential for CARD11-BCL10-MALT1 (CBM) activity, is currently tested in early phase clinical trials. We demonstrated that MALT1 protease inhibition led to the reduction of genes involved in OAsIs resistance, including BCL2A1 (RNA inhibition: 40%, n=6), and induced synergistic cell death in combination with venetoclax (mean synergy score: 20, n=4), irrespective of CARD11 mutational status. Conclusions In summary, our data uncovered control of the mitochondrial apoptosis by CARD11 through BCL2A1 expression. This network has critical implication in the resistance to BTK/BCL2 targeted inhibition. Indeed, CARD11 GOF mutations led not only to BCR-independence and consequently ibrutinib resistance but also to BCL2A1-mediated venetoclax resistance. Nevertheless, our data show that targeting the CBM through MALT1 inhibition reverts this resistance and that MALT1 targeting appears as a promising therapeutic option for counteracting MCL resistance, especially in the context of acquired CARD11 mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Establishment of a Patient-Derived Organoid Culture Platform for Mantle Cell Lymphoma Drug Sensitivity and CAR T Cell Anti-Tumor Activity Assay

Background: With BTK inhibitors and brexucabtagene autoleucel CAR-T cell therapy approved by FDA to treat B-cell malignancies, survival of patients with mantle cell lymphoma (MCL) has been greatly improved. However, frequent therapeutic relapses in patients remains as an unmet medical challenge. Preclinical tumor mouse models such as patient-derived xenografts (PDXs) are often used for drug sensitivity and treatment prediction. However, these conventional cancer models are time-consuming and expensive. In recent years, development and application of patient-derived organoid (PDO) models have led to many discoveries in various types of solid tumors. PDO models have demonstrated many advantages over PDX models. For example, PDOs can be established using biopsies as small as needle biopsies yet with much higher success rates. However, a PDO model has not been reported yet in MCL or in a few other hematologic malignancies. Therefore, in this study, with successful establishment of MCL PDO models, we optimized our protocols and applied this model for drug sensitivity screens and rapid evaluation of immunotherapies. Methods: Patient samples were lysed with RBC lysis buffer and washed with PBS, then cell pellets were resuspended in culture medium containing cytokine cocktails. The cell suspension was aliquoted into V-bottom 96-well plates and spun. The cell aggregates within 50% Matrigel were gently transferred into multiple-well plates as needed. After solidification of the cell-Matrigel mixture, pre-warmed culture medium was added to cover them. The PDOs in 24 or 96-well plates were treated with drug or co-cultured with CAR T cells for 3-5 days. The cell viability and T cell killing activity were determined using CellTiter-Glo 3D assay kits. Results: We successfully established and optimized an organoid culture procedure and culture medium for diverse types of biopsy from patients with MCL. The success rate of MCL organoid development from diverse MCL biopsies was greater than 90%, much higher than for PDX. H&E staining of both tumors and their derived organoids revealed similar histological features (Figure 1A). To examine the drug sensitivity of primary MCL cells, organoid cultures derived from the patient apheresis biopsies that were either BTK inhibitor-sensitive (naive) or multiple drug resistant (to acalabrutinib, rituximab, and venetoclax) were treated with the BTK inhibitors ibrutinib or pirtobrutinib or else doxorubicin (as a control) for 48 h. Cell viability assays indicated that MCL cells in organoid cultures maintained the responses of the original tumor cells to drugs used in clinical treatment. We also demonstrated that the MCL organoids had the same major cell lineage populations as the original biopsy. These results support the notion that the PDO in vitro assay has great potential to evaluate and predict patient responses. In addition, we applied this PDO platform to evaluate CAR T cell anti-tumor activity by co-culturing the primary MCL cells with CD19-targeting CAR T cells in organoids for 3 days. To visualize and quantitate tumor killing potential, the MCL cells were labelled with the green dye 5-chloromethyl fluorescein diacetate. The cocultures were examined by imaging and FACS analysis. Compared to the same batch of peripheral blood monocytes used to generating CAR T cells, the CD19-CAR T cells very effectively eradicated CD19+ MCL cells from the co-cultures (Figure 1B), indicating that the PDO platform is a promising in vitro assay for rapid examining CAR T cell activity. Importantly, we validated drug responses in parallel in PDO and PDX models derived from the same tumors. Our preliminary results displayed high consistency between these assay systems, suggesting that PDO-based assay provides an encouraging platform for predicting clinical treatment response. Conclusion: We have successfully established and optimized an MCL PDO platform that can be used to perform reliable and fast assays for drug sensitivity and CAR T cell anti-tumor activity in vitro. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting EGR1-Medited Metabolic Reprogramming to Overcome Ibrutinib Resistance in Aggressive B Cell Lymphomas

Targeting specific signaling pathways in malignant cells through the use of small-molecule kinase inhibitors has revolutionized the treatment of malignancies. Bruton tyrosine kinase (BTK), a key component of the B cell receptor (BCR) signaling pathway, is a promising molecular target in several types of lymphoma, including diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Ibrutinib, a selective BTK inhibitor, has achieved initial response rates as high as 70% in these lymphomas when used as a single agent. Acquired drug resistance, however, is significant and together with primary resistance makes ibrutinib by itself unsuitable if the goal of treatment is long-term survival. Therefore, understanding and targeting ibrutinib resistance mechanisms is an unmet clinical need. DLBCL, which represents 30% to 40% of newly diagnosed lymphomas, comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). The BCR/BTK signaling pathway is essential for the growth of ABC DLBCL, which is more aggressive and less curable than GCB DLBCL with current regimens. We established two representative ABC DLBCL cell lines (TMD8 and OCI-Ly10) with ibrutinib resistance by gradually increasing the concentration of ibrutinib during passage in culture. RNA-seq analysis demonstrated that the BCR pathway gene signature is enriched in resistant cell lines when compared to parental cells. The most upregulated gene is EGR1, a transcription factor that activates multiple oncogenic pathways including MYC and E2F. Elevated EGR1 expression is also observed in ibrutinib-resistant primary mantle cell lymphoma cells when treated with ibrutinib. Using multiple metabolic and genetic approaches, we discovered that overexpression of EGR1 causes metabolic reprogramming to oxidative phosphorylation (OXPHOS) and ibrutinib resistance. Mechanistically, EGR1 mediates metabolic reprogramming through transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation accelerates intracellular ATP production via the mitochondrial tricarboxylic acid (TCA) cycle, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in patient-derived xenograft mouse models. These findings suggest that targeting EGR1-medited metabolic reprogramming to OXPHOS with IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.