Ibrutinib-resistant Mantle Cell Lymphoma Research Articles

Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma

The use of Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib achieves a remarkable clinical response in mantle cell lymphoma (MCL). Acquired drug resistance, however, is significant and affects long-term survival of MCL patients. Here, we demonstrate that DNA methyltransferase 3A (DNMT3A) is involved in ibrutinib resistance. We find that DNMT3A expression is upregulated upon ibrutinib treatment in ibrutinib-resistant MCL cells. Genetic and pharmacological analyses reveal that DNMT3A mediates ibrutinib resistance independent of its DNA-methylation function. Mechanistically, DNMT3A induces the expression of MYC target genes through interaction with the transcription factors MEF2B and MYC, thus mediating metabolic reprogramming to oxidative phosphorylation (OXPHOS). Targeting DNMT3A with low-dose decitabine inhibits the growth of ibrutinib-resistant lymphoma cells both invitro and in a patient-derived xenograft mouse model. These findings suggest that targeting DNMT3A-mediated metabolic reprogramming to OXPHOS with decitabine provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL.

The Novel CDK9/CDK4/6/PI3K Triple Inhibitor LCI139 for the Treatment of MYC-Driven Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive B cell malignancy with characteristic t(11;14)(q13;q32) translocation juxtaposing IGH and CCND1 gene loci. This chromosomal rearrangement leads to Cyclin D1 overexpression and cell cycle dysregulation at the G1/S phase transition. MCL is generally diagnosed at advanced stages and follows an aggressive course of disease. Although chemotherapy-based combination regimens are initially effective, most patients relapse with a median survival of only 2.5-5 years. While novel targeted therapeutics have shown promising activity in relapsed/refractory patients, inevitable relapse associated with progressively declining efficacy and increasing resistance to single agent targeted therapy is a major concern. To overcome this barrier, we designed in-silico based a novel multitarget small molecule inhibitor LCI139 which simultaneously blocks three oncogenic targets: cyclin-dependent kinase 9 (CDK9), CDK4/6, and phosphatidylinositol-3 kinase (PI3K). LCI139 is a potent CDK9/CDK4/6/PI3K inhibitor with IC 50s of 0.000039µM (CDK9), 0.0015µM/0.0036µM (CDK4/6) 0.070µM (p110α), 0.461µM (p110γ), and 0.214 (p110δ), as determined by cell free assay alpha screens. Dose-dependent screening of LCI139 against a panel of MCL cell lines revealed robust cytotoxic effects with maximal IC 50 of 86.67 nM in JeKo-1 cells, 64.8 nM in Mino cells, and 202.5 nM in Rec-1 cells. Compared with clinical single agent CDK9 inhibitor AZD4573, LCI139 is less toxic to normal human stromal cells (HS-5), whereas both LCI139 and AZD4573 have no significant cytotoxic effect on normal human foreskin fibroblast cells (Hs68). LCI139 treatment at various concentrations for 24hrs induced significant apoptotic response in two MCL cell lines, JeKo-1 (p<0.001 DMSO vs LCI139 100 nM, p<0.0001 DMSO vs LCI139 200 nM) and Mino (p<0.05 DMSO vs LCI139 100 nM, p<0.0001 DMSO vs LCI139 200 nM) as determined by Annexin V/7-AAD apoptosis staining. Similarly, LCI139-induced apoptotic cell death was further confirmed by increasing cl-PARP protein levels compared to control as assessed by Western blot analysis. LCI139 induced robust G2/M cell cycle arrest in JeKo-1 cells and G1 arrest in Mino cells. The mechanism of action of pan-CDK inhibitors has been associated with downregulation of the anti-apoptotic protein MCL-1 through inhibition of transcriptional CDKs. Our results show that short-term exposure of LCI139 (1hr and 3hr) significantly suppressed MCL-1 and MYC mRNA expression in JeKo-1 (p<0.00001 DMSO vs LCI139) and Mino cells (p<0.00001 DMSO vs LCI139). LCI139 suppressed pRpb1 (Ser2), MCL-1 and cMYC protein expression in IgM-stimulated JeKo-1 and Mino cells in a dose-dependent manner. Treatment with LCI139 had no significant effect on pAKT (Ser473) in Jeko-1 cells, whereas pAKT (Ser473) was completely abolished in Mino cells at 500nM of LCI139. Mechanistically, our findings also demonstrate that LCI139 decreased MCL-1 and cMYC protein stability and reduced half-life as accessed by cycloheximide chase assay. This decrease in cMYC stability is associated with decreased of cMYC phosphorylation at Ser62. Adaptive kinome remodeling via drug-induced transcriptional reprogramming is a known contributor to ibrutinib resistant (IR) MCL. RNA-sequencing analysis of JeKo-1 vs Jeko-1 IR cells revealed a total of 3468 differentially expressed genes (FC > 1.5 and FDR < 0.05) where 110 kinases display altered expression in JeKo-1 IR cells. Finally, we show that LCI139 overcomes chronic ibrutinib resistance by decreasing viability in JeKO-1 IR and Mino IR cells with an IC 50 of 79 nM and 89 nM, respectively. Therefore, the multitarget small molecule inhibitor LCI139 has superior potency against IR MCL cell lines and overcomes ibrutinib-resistance at nanomolar doses.

Inhibition of Lysine Acetyltransferases p300/CBP Overcomes BTK Inhibitor Resistance in Mantle Cell Lymphoma Cells

Background: Although Bruton's Tyrosine Kinase inhibitors (BTKis) are effective against relapsed refractory mantle cell lymphoma (MCL), the limited duration of therapeutic response and the emergence of resistance remain clinical issues. We conducted a CRISPR/Cas9 screen to identify the genes responsible for BTKi sensitivity and resistance. Materials and Methods: To identify the genes responsible for BTKi resistance, we used a CRISPR library (Kosuke Yusa, V3), targeting 18,740 human genes. We compared three BTKis, ibrutinib, acalabrutinib and tirabrutinib, using DMSO as a control. We transduced Granta519, an ibrutinib-resistant MCL cell line expressing Cas9 with the CRISPR library lentiviruses. Next, we counted sgRNA copy numbers using next generation sequencing after a two-week drug treatment. We identified 76 genes that sensitized cells to the BTKis upon knockout. After excluding common essential genes based on the Cancer Dependency Map portal (depmap.org/portal/), we ranked these genes according to the extent to which the combination of their knockout and BTKis inhibited cell proliferation. Results: Lysine acetyltransferases (KATs), p300 and CBP were ranked first and ninth, respectively. Knockout of p300 or CBP alone moderately decreased cell numbers, where as their combination with BTKi treatment significantly decreased cell numbers. To further validate this synergistic effect, we performed an MTS assay in combination with p300/CBP inhibitors (C-646 and A-485) and ibrutinib in MCL cell lines (Granta519 and Mino). The p300/CBP inhibitor increased ibrutinib sensitivity, leading to a up to four-fold greater suppression of cell viability. This effect was even greater than that of existing drugs, including venetoclax (a BCL-2 inhibitor), and idelalisib (a PI3K inhibitor), which are thought to have synergistic effects. The synergistic effect of a p300/CBP inhibitor and ibrutinib was also observed in long-term cultures for up to two weeks in both ibrutinib-resistant and ibrutinib-sensitive MCL cell lines. Furthermore, combination treatment resulted in an enhancement of G0/G1 cell cycle arrest and an increase in apoptotic cells in the MCL cell lines. To elucidate the mechanism underlying this synergistic effect, we measured the expression of IκBα mRNA as an indicator of NFκB activity by real-time PCR. In Granta519, ibrutinib alone caused an initial decrease in IκBα mRNA expression followed by a rebound increase. However, the addition of A-485 decreased the rebound and suppressed IκBα mRNA expression. These results indicate that ibrutinib and A-485 cooperatively suppress NFκB activity. The combination of ibrutinib and A-485 synergistically suppressed phosphorylation of PLCγ2 and of AKT as confirmed by Western blotting. This suggests that downstream PLCγ2 and AKT activation might be involved in ibrutinib resistance. Lentiviral overexpression of p300 in Granta519 cells enhanced phosphorylation of AKT. On the other hand, ibrutinib suppressed the expression of p300, and conversely, p300/CBP inhibitors suppressed the expression of BTK. These data suggest that the expression of p300 and BTK are dependent on each other. We hypothesize that direct or indirect interactions may occur through phosphorylation by BTK and acetylation by p300. Conclusion: Our CRISPR screen identified lysine acetyltransferases p300/CBP as BTK inhibitor resistance genes in human MCL cells. The combination of a p300/CBP inhibitor and a BTK inhibitor synergistically suppresses both BTK and AKT, leading to NFκB suppression. Taken together, we first identified that the combination of p300/CBP inhibition and BTK inhibition could be a promising strategy for treating patients with MCL.

PRT2527, a Novel Highly Selective Cyclin-Dependent Kinase 9 (CDK9) Inhibitor, Has Potent Antitumor Activity in Combination with BTK and BCL2 Inhibition in Various Lymphoid Malignancies

CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. We also show that combination of PRT2527 with BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) leads to more robust and durable responses in multiple pre-clinical models. In these studies, the combination of PRT2527 with BTKi or BCL2i was well tolerated. Mechanistically, we show that in cell lines, BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency. Concurrent depletion of MCL1 and BFL1 with PRT2527 led to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In a similar fashion, Venetoclax-driven BCL2 inhibition also potentiated the anti-tumor activity of PRT2527. Consistent with our findings in vitro, we confirmed potent combinatorial inhibition of tumor growth by PRT2527, administered once weekly, and BTKi or BCL2i, administered daily, in CDX and PDX in vivo models of DLBCL and MCL. We observed reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrated enhanced induction of apoptosis by PRT2527 in primary CLL patient samples simultaneously treated with BTKi or BCL2i. Additionally, we demonstrated that PRT2527 potently inhibits growth in Ibrutinib-resistant MCL cell lines. Further work characterizing PRT2527 as monotherapy and in combination with BTKi and BCL2i in in vivo models of BTKi-resistant MCL and CLL is currently ongoing. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory hematologic malignancies.

HARNESSING BTKI THERAPY BY CDK4/6I CONTROL OF T CELL SURVEILLANCE

Introduction: Drug resistance remains a formidable challenge in mantle cell lymphoma (MCL). Cell cycle dysregulation driven by aberrant Cyclin D1 and CDK4 expression is a hallmark for MCL. By inhibition of CDK4/6, we have developed a novel strategy that both inhibits proliferation of MCL cells and reprograms them for cytotoxic killing. In a phase I clinical trial we investigated if inhibition of cyclin D1/CDK4 with palbociclib could reprogram recurrent MCL to deepen and prolong the ibrutinib response, and the mechanism for resistance by longitudinal genomic analysis of sequential samples from individual patients in the context of the clinical response. Methods: Palbociclib was administered to recurrent MCL patients on days 1–21 of a 28-day cycle; ibrutinib was given continuously. For longitudinal genomic analysis, sequential tissue and blood specimens from 27 evaluable MCL patients before, during therapy and on progression were collected. Single cell RNA-seq (scRNA-seq) of PBMC or the monocytic fractions from bone marrow and lymph node was performed using a unique in house MCL-specific library. The data were then subject to multiplex analysis with whole transcriptome sequencing and whole exome sequencing of purified MCL cells and flowcytometry of the same samples in conjunction with IHC. Results: Palbociclib appears to deepen and prolong the ibrutinib response; the CR rate was 42% CR, and 5 patients (2 CR and 3 PR) remained on therapy for >8 years. Longitudinal scRNA-seq further revealed that MCL cells comprise 4 major transcriptomically distinct clusters (C)s. Primary resistance and progression on therapy were associated with a marked expansion of either long-live non-proliferating C3 cells or C2 cells that fuel the proliferating C4 cells. Resistance was associated with a profound reduction in MHC I and MHC II expression and disease progression was further accompanied by a rapid loss of both CD8+ and CD+ T cells. This suggests T cell surveillance plays a critical role in maintaining a durable ibrutinib response. Guided by our discoveries, we have restored ibrutinib sensitivity by target patient-specific expansion of C2 or C3 MCL cells ex vivo. In one patient, this led to restoration of CD4+ and CD8+T cells and a CR over 2.5 years. Conclusion: T cell surveillance plays a critical role in prolonging the ibrutinib response by CDK4/6 inhibition, implicating genome-guided combination therapy to overcome ibrutinib resistance in MCL. The research was funded by: PO1 grant from the National Cancer Institute MCL-RI grant from Leukemia and Lymphoma Society Keywords: combination therapies, genomics, epigenomics, and other-omics, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

IMMUNE‐DEPLETED TUMOR MICROENVIRONMENT IS ASSOCIATED WITH POOR OUTCOMES AND BTK INHIBITOR RESISTANCE IN MANTLE CELL LYMPHOMA

Introduction: An understanding of the tumor microenvironment (TME) and its cellular and analyte composition plays a critical role in promoting mantle cell lymphoma (MCL) cell growth, proliferation. To further understand the relevance of the tumor-immune landscape in tissue microenvironments, we performed multiomic profiling to characterize the TME in tissues from MCL patients and examined the relationship between TME subtypes and their impact on clinical outcome and the response to BTKi. Methods: This cohort study was conducted under an IRB approved protocol for MCL patients at our Center. Tissue biopsies (28 lymph nodes and 13 other tissues) were collected from 41 patients with MCL. We conducted whole exome sequencing (WES; n = 41) and RNA-seq (n = 41) from MCL tissue biopsies from patients treated with BTKi (ibrutinib, acalabrutinib or zanubrutinib) in combination with the analysis of a published MCL cohort (n = 122). Joint WES and RNA-seq mutation calling and TME molecular signatures were categorized based on response to BTKi. All WES and bulk RNA sequencing was performed with Illumina HiSeq4000 using a 76 bp paired end configuration. Results: Unsupervised clustering identified four MCL TME subtypes reflecting distinct tumor-immune cell gene signatures. Four distinct groups—normal lymph node (n = 27), immune cell-enriched (n = 46), mesenchymal (n = 44) and immune cell-depleted “D” (n = 51). “D” subtype was predominant in patients with primary BTKi resistance. A high tumor proliferation gene signature was observed exclusively in the “D” group. We further observed that Ki-67% from tissue biopsies had a linear correlation with proliferation rate signature genes and significantly overexpressed in the D group (p = 0.002). Somatic mutations such as TP53, NSD2, NOTCH1, KMT2D, SMARCA4, which were previously reported in ibrutinib-resistant MCL and/or in refractory high-risk MCL patients, were predominant in the Dsubtype. Finally, the evaluable patients (n = 39) were divided according to response to BTKi- sensitive, primary resistant and acquired resistant and MFP clusters. Patients with primary and acquired resistance had significant proportion of patients with D subtype (p = 0.004), compared to N and IE subtypes. Primary and BTKi resistant patients had a trend of inferior survival compared to sensitive patients (p = 0.07). Furthermore, we demonstrated that the D TME group had worse overall survival compared to other TME categories (p = 0.001). Keyword: microenvironment The research was funded by: Boston Gene Corp

CTPS1 acts as a prognostic biomarker and therapeutic target in mantle cell lymphoma

Introduction: Mantle cell lymphoma (MCL) represents 6 to 8% of non-Hodgkin’s lymphoma with generally poor prognosis. Cytidine triphosphate synthase 1 (CTPS1) is a critical regulatory enzyme in cytidine metabolism, catalyzing the rate-limiting step in de novo CTP synthesis. Herein, we aim to investigate the clinical significance and functional mechanisms of CTPS1 in MCL patients. Methods: Lymph node, bone marrow biopsy, peripheral blood specimens and MCL cell lines were utilized as models. Liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based analysis was performed to measure the steady-state level of metabolites. GSE93291 dataset and immunohistochemistry (IHC) staining were utilized to determine the relationship between expression levels of CTPS1 and patient outcome. CRISPR/Cas9 editing was employed to engineer the loss-of-function models of CTPS1 and other key enzymes involved in CTP synthesis. Results: Metabolic Profiling revealed dramatically aberrant cellular abundance of metabolic intermediates in the CTP synthesis pathway (Figure 1A). For GSE93291 analysis, a nucleotide metabolism related prognostic model was established by bioinformatic analysis and CTPS1 was screened out by highest regression coefficient (Figure 1B). IHC staining of CTPS1 in 105 MCL tissues confirmed that elevated CTPS1 expression was significantly associated with poor PFS (P = 0.038) and OS (P = 0.001) in MCL patients (Figure 1C-D). Multivariate Cox regression analysis showed that high CTPS1 expression was an independent prognostic indicator of both PFS and OS. CTPS1 depletion significantly impaired outgrowth and increased apoptosis of MCL cells, suggesting that CTPS1 is the major isozyme important for MCL survival (Figure 1E-F). In primary tumor samples, MYC protein levels were significantly correlated with CTPS1 protein levels (P < 0.001, R = 0.638). MYC knockout blocked CTPS1 mRNA and protein expression prior to induction of cell death, suggesting a requisite role of MYC in CTPS1 transcription regulation (Figure 1G). Remarkably, TP53 aberrant and ibrutinib-resistant MCL cells rely on cytidine metabolism (Figure 1H). Genetic or chemical inhibition (3-deazauridine) of CTPS1 reversed poor responsiveness of MCL cell lines to ibrutinib (Figure 1I). Cytidine level was significantly increased in TP53-deficient MCL cells and the effect of CTPS1 inhibition was independent of TP53 status (Figure 1K). Gene set enrichment analysis of RNA-sequencing revealed that CTPS1 inhibition induced DNA damage and apoptosis. Moreover, CTPS1 inhibition caused cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) activation, which triggered innate immune pathway, leading to cells growth inhibition (Figure 1L). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Diagnostic and Prognostic Biomarkers, Metabolism No conflicts of interests pertinent to the abstract.

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.

Abstract 5974: Synergistic activity of the CDK4/6 antagonist narazaciclib (ON123300) with irreversible BTK inhibition in ibrutinib-resistant mantle cell lymphoma

Abstract Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of B-cell lymphoma including the aggressive mantle cell lymphoma (MCL) subtype, although primary and acquired resistance is frequently observed. Interestingly, preliminary results have suggested that the second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor, ON123300 (narazaciclib), may be effective in preclinical models of BTKi-resistant MCL. We compared the efficacy and safety of ON123300 vs the approved CDK inhibitors palbociclib, abemaciclib or ribociclib, in association with various BTKi, in a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib, or the second generation acalabrutinib. We evaluated the effects of these combinations by CellTiter-Glo proliferation assay, FACS-mediated analysis of cell cycle and apoptosis, RT-PCR and western blot. Efficacy and safety of ON123300 in vivo was evaluated in a chicken embryo chorioallantoic membrane (CAM) xenograft model of MCL. We observed that ON123300 exhibited a significant antitumor activity in MCL cell lines independently of their sensitivity to ibrutinib, with IC50 at 72h ranging from 0.7 to 7.1µM (mean: 3.61±2.1µM), while it achieved a 32% tumor growth inhibition, with no detectable toxicity, in the CAM-MCL model. In vitro, ON123300 demonstrated a superior activity than palbociclib and ribociclib, and a similar effect that abemaciclib. Interestingly, ON123300 exhibited an antitumor activity comparable to ibrutinib but was more effective than acalabrutinib. ON123300 single agent treatment repressed several positive regulators of the G2/M cell cycle phase and triggered the accumulation of the CDK inhibitors p21, p16, and phospho-p27, with the consequent loss of phospho-Histone H3 and phospho-CDK2. Accordingly, ON123300 treatment evoked a 20-35% increase in G1 cell cycle fraction at 24h, which preceded the ignition of mitochondrial apoptosis. When combined with ibrutinib, rather than with acalabrutinib, ON123300 exhibited a significant antitumor activity with synergistic combination indexes in both BTK-sensitive and BTK-resistant cell lines. This phenomenon was associated with a slight but constant (+10-15%) augmentation of G1 blockade, histone H3/CDK2 dephosphorylation and p27/p16 accumulation in cells exposed to ibrutinib-ON123300 combination, with no detectable potentiation of apoptosis. In conclusion, ON123300 is safe and effective as single agent in preclinical models of MCL, including BTKi-resistant cases. Due to its complete distinct MoA from BTKi involving the direct modulation of cell cycle, ON123300, but not abemaciclib or palbociclib, can achieve significant synergistic activity in combination with ibrutinib, and in lower extent with acalabrutinib, especially in BTKi-resistant MCL cases. Citation Format: Nuria Profitos-Peleja, Marcelo L. Ribeiro, Adar Makovski Silverstein, Stephen Cosenza, Gaël Roué. Synergistic activity of the CDK4/6 antagonist narazaciclib (ON123300) with irreversible BTK inhibition in ibrutinib-resistant mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5974.

Abstract 6208: Targeting DNA polymerase theta and ATM leads to synergistic killing of mantle cell lymphoma cells

Abstract Introduction: Rapid cell proliferation requires intact and faithful DNA damage repair mechanisms. DNA polymerase theta (POLQ) plays a key role in repairing DNA double-strand breaks through the microhomology-mediated end-joining (MMEJ), which is one of the three main pathways involved in repairing replication-induced double-strand breaks. Limited data have suggested that concurrent depletion of POLQ and ataxia-telangiectasia mutated (ATM) could be embryonic lethal. Hence, this phenomenon has the potential to be exploited for therapeutic benefit in cancers where ATM mutations are commonly seen. Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma marked by (11;14) translocation with ATM alterations seen in 40-50% of patients. There is an unmet need to find novel therapeutic strategies, especially in relapsed and/or refractory (R/R) MCL. Here, we investigated whether targeting POLQ and ATM could be a potential therapeutic strategy in MCL. Methods: In vitro studies were conducted by using MCL cell lines. CRISPR-Cas9 system was used to genetically deplete POLQ and ATM genes and sgRNAs co-expressing fluorescence markers were used to track the cell population with respective genotypes over time. Cell viability was assessed by CellTiter-Glo assay and flow cytometry. All cell lines were profiled for ATM expression and activity. A p-value of &amp;lt; 0.05 was considered statistically significant. The combination index of &amp;lt;1 was defined as synergistic. Results: CRISPR-Cas9-mediated depletion of POLQ significantly decreased cell proliferation in multiple MCL cell lines. In particular, Granta-519, which possesses a single copy of kinase-dead ATM that is reduced in expression, was most sensitive to POLQ depletion. Concurrent genetic depletion of ATM and POLQ resulted in a synergistic antiproliferative effect in ATM-proficient MCL cell lines. Subsequently, this cellular phenotype caused by the genetic intervention was recapitulated by using two POLQ inhibitors (novobiocin and ART558) and an ATM inhibitor (AZD0156). In vitro, single-agent treatment with novobiocin or ART558 caused a significant cytotoxic effect at physiologically relevant concentrations in ATM-deficient cells and co-treatment of novobiocin or ART558 with AZD0156 was synergistic in killing ATM-proficient MCL cells. Importantly, POLQ inhibitors significantly decreased the cell viability of MCIR1, which is an ibrutinib-resistant MCL cell line. Mechanistically, novobiocin or ART558 treatment induced gH2AX and cleaved PARP upregulation, which was further enhanced by ATM depletion, suggesting that co-inhibition of POLQ and ATM caused apoptosis due to the accumulation of unrepaired DNA damage. Conclusion: POLQ is a promising target in MCL, especially in ATM-deficient setting. In ATM-proficient MCL, targeting ATM and POLQ is synergistic. Our data has the potential to uncover novel biomarker-driven drug therapy of POLQ inhibitors in R/R MCL. Citation Format: Jithma Prasad Abeykoon, Shuhei Asada, Kalindi Parmar, Xiaosheng Wu, Thomas Witzig, Geoffrey Shapiro, Alan D. D'Andrea. Targeting DNA polymerase theta and ATM leads to synergistic killing of mantle cell lymphoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6208.

Abstract 3451: XS-04: A first-in-class dual IRAK4/BTK inhibitor for the treatment of hematological malignancies

Abstract Bruton's tyrosine kinase (BTK), a critical mediator of B-cell receptor (BCR) signaling, is a validated therapeutic target found across multiple B-cell cancers. Though initially responding to treatment with a BTK inhibitor, many patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), or Waldenström macroglobulinemia (WM) eventually progressed and clinical responses to BTK inhibitors are often poor in diffuse large B-cell lymphoma (DLBCL) patients. These highlight the need to understand the resistance mechanisms and improve responses to BTK inhibitors. The serine/threonine protein kinase IRAK4 is a key modulator of MYD88, which is found to be mutated in various aggressive B-cell lymphomas, including DLBCL. As BCR signaling and MYD88 signaling converge downstream on NF-kB activation, which plays an essential role in proliferation and survival of lymphoma cells, blockade of the two signaling pathways presents a potential therapeutic strategy. Here, we investigated the pharmacokinetic properties and anti-tumor activity of XS-04, a first-in-class dual inhibitor targeting both IRAK4 and BTK, using preclinical models of DLBCL subtypes, including tumors resistant to BTK inhibitors. XS-04 showed potent inhibition of IRAK4 and BTK kinases, with IC50 values of 0.2 and 2.9 nM, respectively. Favorable pharmacokinetic properties were also seen across multiple non-clinical species. Using in vitro anti-proliferative assays, we found that XS-04 inhibited proliferation of various DLBCL cell lines with IC50s of 30 to 40 nM. Mechanistically, XS-04 suppressed NF-kB activity in DLBCL cells and in vivo xenograft tumors, as indicated by decreased phosphorylation of IkBa. Oral dosing of XS-04 in TMD8 xenograft model led to a dose-dependent inhibition of tumor growth, which was comparable to the inhibition shown by a combination of BTK and IRAK4 inhibitors. In addition, XS-04 treatment resulted in tumor regression in a patient-derived ibrutinib-resistant MCL xenograft model, without significant changes in body weight. XS-04, a first-in-class IRAK4/BTK dual inhibitor, shows broad anti-tumor activity across preclinical models of DLBCL subtypes, including tumors resistant to BTK inhibitors. These results support XS-04 as a therapeutic candidate for the treatment of hematological malignancies. An IND submission is planned for 2023. Citation Format: Chunyan Zhao, Jianfei Wang, Aiguo Liu, Xin Gao, Yanfen Teng, Lei Liu, Yang Zhang, Xiaohong Yu. XS-04: A first-in-class dual IRAK4/BTK inhibitor for the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3451.

Cotargeting of BTK and MALT1 overcomes resistance to BTK inhibitors in mantle cell lymphoma.

Bruton's tyrosine kinase (BTK) is a proven target in mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma. However, resistance to BTK inhibitors is a major clinical challenge. We here report that MALT1 is one of the top overexpressed genes in ibrutinib-resistant MCL cells, while expression of CARD11, which is upstream of MALT1, is decreased. MALT1 genetic knockout or inhibition produced dramatic defects in MCL cell growth regardless of ibrutinib sensitivity. Conversely, CARD11-knockout cells showed antitumor effects only in ibrutinib-sensitive cells, suggesting that MALT1 overexpression could drive ibrutinib resistance via bypassing BTK/CARD11 signaling. Additionally, BTK knockdown and MALT1 knockout markedly impaired MCL tumor migration and dissemination, and MALT1 pharmacological inhibition decreased MCL cell viability, adhesion, and migration by suppressing NF-κB, PI3K/AKT/mTOR, and integrin signaling. Importantly, cotargeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in ibrutinib-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that MALT1 overexpression associates with resistance to BTK inhibitors in MCL, targeting abnormal MALT1 activity could be a promising therapeutic strategy to overcome BTK inhibitor resistance, and cotargeting of MALT1 and BTK should improve MCL treatment efficacy and durability as well as patient outcomes.

Targeting glutaminase is therapeutically effective in ibrutinib-resistant mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by frequent relapses. The development of resistance to ibrutinib therapy remains a major challenge in MCL. We previously showed that glutaminolysis is associated with resistance to ibrutinib. In this study, we confirmed that glutaminase (GLS), the first enzyme in glutaminolysis, is overexpressed in ibrutinib-resistant MCL cells, and that its expression correlates well with elevated glutamine dependency and glutaminolysis. Furthermore, we discovered that GLS expression correlates with MYC expression and the functioning of the glutamine transporter ASCT2. Depletion of glutamine or GLS significantly reduced cell growth, while GLS overexpression enhanced glutamine dependency and ibrutinib resistance. Consistent with this, GLS inhibition by its specific inhibitor telaglenastat suppressed MCL cell growth both in vitro and in vivo. Moreover, telaglenastat showed anti-MCL synergy when combined with ibrutinib or venetoclax in vitro, which was confirmed using an MCL patient-derived xenograft model. Our study provides the first evidence that targeting GLS with telaglenastat, alone or in combination with ibrutinib or venetoclax, is a promising strategy to overcome ibrutinib resistance 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

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.

Inhibition of LINK-A lncRNA overcomes ibrutinib resistance in mantle cell lymphoma by regulating Akt/Bcl2 pathway.

Ibrutinib, a bruton tyrosine kinase (BTK) inhibitor which suppresses B-cell receptor signaling, has remarkably improved the outcome of patients with mantle cell lymphoma (MCL). However, approximately 33% of MCL patients have primary Ibrutinib resistance, and acquired Ibrutinib resistance is nearly universal. Long intergenic non-coding RNA for kinase activation (LINK-A) exerts oncogenic role in different types of tumors, but the role of LINK-A in intrinsic ibrutinib resistance in MCL is still unclear. Here, LINK-A expression level was first assessed using quantitative Real-time PCR (qPCR) and immunofluorescence analysis in five MCL cell lines. The effect of LINK-A on regulating MCL cells viability and apoptosis was assayed using CCK-8 and TdT-mediated dUTP nick end labeling (TUNEL) assay, respectively. The association of LINK-A with AKT activation and B cell lymphoma 2 (Bcl2)expression was evaluated using qPCR and western blot analysis. We found that LINK-A level was elevated in Ibrutinib-resistant MCL cell lines (Mino, REC-1, MAVER-1, and Granta-519) compared to Ibrutinib-sensitive MCL cell lines (Jeko-1). Functionally, LINK-A overexpression in Jeko-1 cells enhanced cell viability and repressed Ibrutinib-induced cell apoptosis. LINK-A knockdown in MAVER-1 cells decreased cell viability and further accelerated Ibrutinib-induced cell apoptosis. LINK-A overexpression enhanced Bcl2 expression in Jeko-1 cells, and Bcl2 inhibition blocked the effect of LINK-A on increasing cell viability in the presence of Ibrutinib. On the contrary, LINK-A knockdown reduced Bcl2 expression in MAVER-1 cells, and Bcl2 overexpression damaged the role of LINK-A inhibition in regulating cell viability. Mechanistically, LINK-A positively regulated the activation of AKT signaling, and inhibition of AKT signaling destroyed LINK-A-induced increased of Bcl2 and resulted in a subsequent suppression of cell viability. Taken together, the current results demonstrate that LINK-A inhibition overcomes Ibrutinib resistance in MCL cells by regulating AKT/Bcl2 pathway.

PRMT5 Inhibition Modulates E2F1 and P53 to Restore Cell Cycle Regulation and Drive DNA Damage Response in Ibrutinib-Resistant Mantle Cell Lymphoma

Introduction:Ibrutinib is widely used for relapsed/refractory mantle cell lymphoma (MCL), however nearly 1/3 of patients have primary resistance and the remaining patients will inevitably acquire resistance with poor overall survival. There is an urgent need for novel therapies targeting pro-survival signaling pathways triggered by ibrutinib resistance. Pharmacologic inhibition of Protein Arginine Methyltransferase 5 (PRMT5) represents a novel therapeutic approach to overcome ibrutinib resistant MCL (IR-MCL). PRMT5 has many essential functions in normal and malignant B cells, however in the context of IR-MCL the mechanisms of PRMT5 inhibitor mediated cell death have not been described. Genomic co-deletions of MTAP/CDKN2A are commonly detected in IR-MCL cells. MTAP deletion and subsequent accumulation of the metabolite MTA, sensitizes cells to further inhibition of PRMT5. Although the published literature defines this therapeutic rationale for targeting PRMT5 in cancers with co-deletion of MTAP/CDKN2A, the literature lacks studies specifically targeting PRMT5 in IR-MCL.Methods:Here we report the primary mechanism of PRMT5 inhibitor mediated cell death in the context of IR-MCL, utilizing a combination of Whole Exome Sequencing (WES), RNA-seq, protein expression, and cell cycle analysis (PI-DNA staining with flow cytometry) in 8 MCL cell lines and 3 patient derived xenografts (PDXs) of IR-MCL. Pharmacologic inhibition of PRMT5 was performed with two selective compounds: PRT-382 and C220 (Prelude Therapeutics). Inducible CRISPR/CAS9-gRNA was used to selectively knock out MTAP and CDKN2A.Results:Comparing MCL cell lines with primary ibrutinib resistance, we found an inverse correlation between PRT-382 ic50 and ibrutinib ic50 (Spearman: r = -0.94, p = 0.017). In MCL cell lines, low expression of MTAP also correlated with lower global Symmetric Di-Methyl Arginine (SDMA) and enhanced sensitivity to C220 respectively (Pearson: r = 0.84, p = 0.01; r = 0.87, p = 0.01). Genomic deletion of MTAP in a cell line with primary ibrutinib resistance (Jeko MTAP-WT) enhanced sensitivity to PRMT5i, whereas MTAP knock-in (KI) in CCMCL, an MTAP-null cell line, decreased sensitivity to PRMT5i. The treatment of Jeko-MTAP-KI and CCMCL-MTAP-KO with MTA, further confirmed that genomic deletion of MTAP confers enhanced vulnerability to PRMT5i in MCL cell lines.In a PDX generated in our lab from an MCL patient with acquired IR, treatment with PRT-382 decreased tumor infiltration of multiple organs and ki-67 compared to ibrutinib or control (H&E and IHC staining). To mechanistically address the nature of the anti-tumor activity of PRMT5i in IR-MCL, we treated PDX mice with PRT-382 or vehicle control for 2 weeks to obtain ex-vivo samples for NGS methods. Splenic MCL tumor cells (Hu-CD19+) where subjected to transcriptomic profiling by RNA-seq (n=3 per Tx group). RNA-seq revealed 1014 down and 1124 up differentially expressed genes (DEGs) (q < 0.01, |log2FC| > 0.05). Gene Set Enrichment Analysis (GSEA) identified activation of the p53 pathway (NES = 1.73), with repression of E2F targets, G2M checkpoints, Epithelial to Mesenchymal Transition (EMT), and PRMT5 targets among the top gene sets modulated with PRMT5i (NES = -2.51, -2.26, -2.06, -1.79). Comparing genomic alterations across MCL cell lines and 3 PDXs of IR-MCL, co-deletion of MTAP/CDKN2A and/or a functional/WT copy of TP53 defines a therapeutic vulnerability to PRMT5i in 9 out of 11 cases (82%). In MCL, PRMT5i resulted in p53-dependent G1/S cell cycle arrest and p53-dependent DNA damage induced apoptosis by transcriptional activation of the p53 target genes (CDKN1A, BBC3, BAX, PHLDA3, andSESN1). Western blot analysis confirmed E2F target gene repression (TK1, CCNA2, CHK1, CDK1) and accumulation of DNA damage response proteins (cleaved PARP, cleaved CASP3, p-H2AX) that are further enhanced with combination of doxorubicin.Conclusion:Pharmacologic inhibition of PRMT5 with the clinical-grade, novel small molecule inhibitor (PRT543, Prelude Therapeutics) merits further investigation in ongoing clinical trials (NCT03886831, clinicaltrials.gov), specifically for the treatment of IR-MCL with deletion of MTAP and prior to the genomic mutation of TP53. We are currently exploring novel combinations to maximize the therapeutic potential of PRMT5 inhibition in this disease. DisclosuresByrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees. Vaddi: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

Immune-Depleted Tumor Microenvironment Signature Is Associated with BTK Inhibitor Resistance in Mantle Cell Lymphoma

Immune-Depleted Tumor Microenvironment Signature Is Associated with BTK Inhibitor Resistance in Mantle Cell Lymphoma

MiRNA-223-3p modulates ibrutinib resistance through regulation of the CHUK/Nf-κb signaling pathway in mantle cell lymphoma

Since the use of Bruton's tyrosine kinase (BTK) inhibitor ibrutinib in relapsed/refractory (R/R) mantle cell lymphoma (MCL), the problem of drug resistance has become increasingly prominent. Though it has been proven that the nonclassic nuclear factor κB pathway (nonclassic NF-κB pathway) correlates with ibrutinib resistance in MCL, the upstream regulator is unknown. In the present study, conserved helix-loop-helix ubiquitous kinase (CHUK) overexpression accelerated proliferation and suppressed apoptosis of MCL cells after ibrutinib treatment in vitro. The results of luciferase reporter assay, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot revealed that CHUK was targeted and negatively regulated by miRNA-223-3p. miRNA-223-3p knockdown promoted proliferation and inhibited apoptosis of MCL cells after ibrutinib treatment in vitro and vivo, whereas CHUK knockdown reversed downregulated miRNA-223-3p-promoted cell proliferation after ibrutinib treatment in vitro. In conclusion, miRNA-223-3p modulates ibrutinib resistance through regulation of the CHUK/NF-κB signaling pathway in MCL, which is crucial in providing a marker to predict disease response.

MCIR1: A patient‐derived mantle cell lymphoma line for discovering new treatments for ibrutinib resistance

Despite the unprecedented success of ibrutinib in lymphoma therapy, the development of ibrutinib resistance due to acquired BTK or PLCγ2 mutations has become a new clinical problem. However, not all resistance is mediated by these mutations and these mechanisms are poorly understood due to a lack of study tools that truly recapitulate this clinical scenario. We established a novel patient-derived ibrutinib-resistant mantle cell lymphoma (MCL) line named MCIR1. Using immunological, molecular, and cytogenetic approaches, we comprehensively characterized MCIR1 and further demonstrated its utility in the study of resistance mechanisms and treatments to overcome this resistance. We show that MCIR1 is a bona fide ibrutinib-resistant MCL cell line with normal BTK-/PLCγ2 but ibrutinib-resistant ERK1/2 and AKT1 signaling. RNA-Seq analysis revealed a robust non-canonical NF-kB signaling that drives the ibrutinib resistance. We also demonstrate the potential utility of a MCIR1-based cell and mouse model for the discovery of new treatments to overcome BTK inhibitor resistance. We have established the first patient-derived ibrutinib-resistant MCL cell line MCIR1 that lacks BTK or PLCγ2 mutations but exhibits a hyperactive non-canonical NF-kB pathway. We further demonstrate its utility in the discovery and validation of new drugs to overcome this resistance.