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AMPK-mediated PRPS1/2 phosphorylation copes with energy stress in tumorigenesis.

Supplementary figure legends and Supplementary Table S1.

Purpose: The mammalian target of rapamycin (mTOR) kinase acts downstream of phosphoinositide 3-kinase/Akt to regulate cellular growth, metabolism, and cytoskeleton. Because ∼60% of sporadic colorectal cancers (CRC) exhibit high levels of activated Akt, we determined whether downstream mTOR signaling pathway components are overexpressed and activated in CRCs.Experimental Design: HCT116, KM20, Caco-2, and SW480 human CRC cells were used to determine the effects of pharmacologic (using rapamycin) or genetic (using RNAi) blockade of mTOR signaling on cell proliferation, apoptosis, cell cycle progression, and subcutaneous growth in vivo.Results: We show that the mTOR complex proteins mTOR, Raptor, and Rictor are overexpressed in CRC. Treatment with rapamycin significantly decreased proliferation of certain CRC cell lines (rapamycin sensitive), whereas other cell lines were resistant to its effects (rapamycin resistant). Transient siRNA-mediated knockdown of the mTORC2 protein, Rictor, significantly decreased proliferation of both rapamycin-sensitive and rapamycin-resistant CRC cells. Stable shRNA-mediated knockdown of both mTORC1 and mTORC2 decreased proliferation, increased apoptosis, and attenuated cell cycle progression in rapamycin-sensitive CRCs. Moreover, stable knockdown of both mTORC1 and mTORC2 decreased proliferation and attenuated cell cycle progression, whereas only mTORC2 knockdown increased apoptosis in rapamycin-resistant CRCs. Finally, knockdown of both mTORC1 and mTORC2 inhibited growth of rapamycin-sensitive and rapamycin-resistant CRCs in vivo when implanted as tumor xenografts.Conclusions: Targeted inhibition of the mTORC2 protein, Rictor, leads to growth inhibition and induces apoptosis in both rapamycin-sensitive and rapamycin-resistant CRCs, suggesting that selective targeting of mTORC2 may represent a novel therapeutic strategy for treatment of CRC.(Clin Cancer Res 2009;15(23):7207–16)

Supplementary Data from Targeted Inhibition of Mammalian Target of Rapamycin Signaling Inhibits Tumorigenesis of Colorectal Cancer

Bruton’s tyrosine kinase (BTK) is a great target in mantle cell lymphoma (MCL). This is evident by multiple FDA approvals of covalent BTK inhibitors (BTKi, e.g. ibrutinib, acalabrutinib and zanubrutinib) and recent exciting clinical data on non-covalent BTKi pirtobrutinib. However, resistance to BTKi is a major clinical challenge and the resistance mechanism is not yet fully understood. To address this, we generated JeKo BTK KD cells via BTK knockdown (KD) through by CRISPR Cas9 in BTKi-sensitive JeKo-1 cells, which resulted in superior resistance to BTKi and cell growth defects in vitro. Interestingly, JeKo BTK KD cells demonstrated early tumor cell engraftment and growth in subcutaneous xenograft models, while parental JeKo-1 cells showed much later engraftment but with much faster growth kinetics. To understand this and the BTKi-resistance mechanism, we first performed bulk RNA sequencing analysis and identified MALT1, but not its well-known binding partners CARD11 and BCL10, as one of the top overexpressed genes in BTKi-resistant MCL cells, including JeKo BTK KD cells. Genetic knockout (KO) of MALT1 or CARD11 by CRISPR Cas9 in JeKo-1 resulted in defects in cell growth in vitro and delayed tumor engraftment and growth in vivo. In contrast, MALT1 KO, but not CARD11 KO, in JeKo BTK KD cells remarkably suppressed cell growth in vitro, and tumor engraftment and growth in vivo. These data demonstrate that MALT1 overexpression can drive ibrutinib resistance via bypassing BTK-CARD11 signaling. BTKi-resistant cells including JeKo BTK KD cells showed much higher potency in adhesion to extracellular matrix or stromal cells compared to BTKi-sensitive cells. BTK inhibition or MALT1 inhibition significantly suppressed cell adhesion and migration to extracellular matrix or stromal cells. Furthermore, BTK KD and MALT1 KO but not CARD11 KO in JeKo-1 cells remarkably suppressed tumor cell dissemination and growth in spleen, liver, bone marrow and peripheral blood. MALT1 KO in JeKo BTK KD cells further suppressed tumor cell dissemination. Consistent with this, MALT1 inhibition greatly suppressed tumor cell dissemination and growth in spleen, bone marrow and peripheral blood of an ibrutinib-resistant patient-derived xenograft model. Therefore, both BTK and MALT1 are critical for tumor cell adhesion and dissemination in vivo in a CARD11-independent manner. Furthermore, co-targeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in BTKi-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that (1) BTK and MALT1 are key molecules that control MCL cell growth and dissemination, (2) MALT1 overexpression drives resistance to BTKi in MCL, (3) targeting MALT1 is a promising therapeutic strategy to overcome BTKi resistance, and (4) co-targeting of BTK and MALT1 improves efficacy and durability beyond single agents. Citation Format: Vivian Changying Jiang, Yang Liu, Qingsong Cai, Joseph M. McIntosh, Yijing Li, Zhihong Chen, Heng-Huan Lee, Wei Wang, Yixin Yao, Lei Nie, Michael Wang. BTK and MALT1 are critical for cell adhesion and dissemination in 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 3914.

Abstract Inevitable relapses remain as the major therapeutic challenge in patients with mantle cell lymphoma (MCL) despite FDA approval of multiple targeted therapies and immunotherapies. Fc gamma receptors (FcγRs) play important roles in regulating antibody-mediated immunity. FcγRIIB, the unique immune-checkpoint inhibitory member of the FcγR family, has been implicated in immune cell desensitization and tumor cell resistance to the anti-CD20 antibody rituximab and other antibody-mediated immunotherapies; however, little is known about its expression and its immune-modulatory function in patients with aggressive MCL, especially those with multi-resistance. In this study, we found that FcγRIIB was ubiquitously expressed in both MCL cell lines and primary patient samples. FcγRIIB expression is significantly higher in CAR T-relapsed patient samples (p p = 0.05) and rituximab-venetoclax (p = 0.02), but not the rituximab-CHOP combination in JeKo-1 cell line-derived xenograft models. In patient-derived xenograft (PDX) models, BI-1206, as a single agent, showed high potency (p R2 therapy). BI-1206 sensitized the efficacy of rituximab monotherapy in a PDX model with triple resistance to rituximab, ibrutinib and CAR T-therapies (p = 0.030). Moreover, BI-1206 significantly enhanced the efficacy of the rituximab-venetoclax combination (p Graphical Abstract

Despite the fact that pesticides help increase food production, widespread application of pesticides has resulted in negative impact in the environment and human health. Routine and comprehensive screening of pesticides in food and water is important for regulatory agencies to ensure that concentrations of toxic pesticides are below maximum allowable levels. Regardless of the pesticides that are not GC amenable, GC-MS still dominates the analysis of pesticides. The focus of the current chapter is a step-by-step method for GC-MS approaches in analysis of several classes of pesticides, including organochlorines, organophosphates, and triazines. GC-MS is superior or at least equivalent to LC-MS method and derivatization is not required prior to instrumental analysis.

Despite the fact that pesticides help increase food production, widespread application of pesticides has resulted in negative impact in the environment and human health. Routine and comprehensive screening of pesticides in food and water is important for regulatory agencies to ensure that concentrations of toxic pesticides are below maximum allowable levels. Regardless of the pesticides that are not GC amenable, GC-MS still dominates the analysis of pesticides. The focus of the current chapter is a step-by-step method for GC-MS approaches in analysis of several classes of pesticides, including organochlorines, organophosphates, and triazines. GC-MS is superior or at least equivalent to LC-MS method and derivatization is not required prior to instrumental analysis.

Introduction Clinical relapse following CD19 CAR T therapy after failure to BTK inhibitors (BTKi) is a novel and fast growing medical challenge in treating patients with mantle cell lymphoma (MCL). Thus, developing a novel therapy to overcome this BTKi-CAR T dual resistance (Dual-R) is an urgent need. Our unpublished data revealed MYC targets and cyclin-dependent kinase 9 (CDK9) are highly upregulated in the Dual-R compared to BTKi-R samples. CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex. Its inhibition induces acute decline of short-lived mRNA and proteins, especially MYC and MCL-1, in acute myeloid leukemia and diffuse large B-cell lymphoma. Therefore, we hypothesize that targeting CDK9 may turn off MYC-driven tumor survival and drug resistance. BAY-1251152 is a novel selective CDK9 inhibitor with nanomolar potency. However, whether it has the potential to overcome BTKi-CAR T dual resistance has not been assessed. Methods To validate the correlation of MYC and CDK9 with drug resistance and clinical outcome, we performed whole transcriptomic profiling using primary patient samples. To assess the in vitro efficacy of BAY-1251152, we performed cell viability assay and cell apoptosis assay using MCL cell lines and primary patient samples and followed up with functional studies. furthermore, we assessed its in vivo efficacy using patient-derived xenograft (PDX) models derived from MCL patients including one with Dual-R. Results The expression of MYC oncogene associates with ibrutinib resistance and Dual-R, and poor clinical outcome. CDK9 expression does not correlates with ibrutinib resistance but it does associate with Dual-R and poor clinical outcome in CAR T-relapsed patients. CDK9 inhibition by BAY-1251152 is highly potent in anti-MCL activity in MCL cell lines with low nanomolar range of IC50 (59.6-172.3 nM) by inducing robust cell apoptosis. BAY-1251152 induces dose and time-dependent CDK9 inhibition. A rapid decline of phosphorylation of RNA polymerase II, MYC, MCL-1, and Cyclin D1 can be observed as early as 2 hours. Furthermore, BAY-1251152 (10mg/kg, QW) significantly inhibited tumor growth (p = 0.000003) in a PDX model derived from a Dual-R patient without causing apparent toxicity in NSG mice. In addition, BAY-1251152 also significantly suppressed tumor growth in the PDX models derived from a BTKi-R patient (p=0.00015) and a BTKi-venetoclax dual-resistant patient (p=0.009). Conclusion Our findings showed that targeting CDK9 by its specific inhibitor BAY-1251152 led to potent in vitro anti-MCL activity. BAY-1251152 induces fast CDK9 inhibition and rapid decline of MYC, MCL-1 and Cyclin D1 to induce robust cell death. BAY-1251152 is also potent in inhibiting tumor growth in PDX models. These data support that CDK9 is a promising target to overcome BTKi-CAR T dual resistance in MCL, which is in urgent need. Citation Format: Vivian Changying Jiang, Lingzhi Li, Alexa Jordan, Yu Xue, Fangfang Yan, Joseph McIntosh, Yang Liu, Yuxuan Che, Yijing Li, Qingsong Cai, Angela Leeming, Lukas Simon, Zhongming Zhao, Jia Zhou, Michael Wang. Targeting transcription elongation via CDK9 in mantle cell lymphoma patients with dual resistance to BTK inhibition and CAR T therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3955.

Background Due to excellent clinical response, brexucabtagene autoleucel (BA), an anti-CD19 chimeric antigen receptor T (CAR T) cell therapy, was approved by FDA to treat patients with MCL. However, outcomes of BA-relapsed patients are very poor. Therefore, in this study, we performed multi-omics profiling for BA-treated patients and explore the potential mechanisms of response/resistance to BA therapy in MCL. Methods We performed single cell transcriptomic profiling for longitudinally collected primary patient samples (n = 39) from 15 patients (1 refractory, 14 CR and 5 relapsed). Cytokine multiplex analysis of 80 analytes were performed for longitudinal patient plasma samples (n = 80) for 18 patients (17 CR, 5 relapsed and 1 refractory). 15 patients had data available for both analyses. Functional studies were performed to further validate the findings from multi-omic analysis. Results Using single cell RNA sequencing, we demonstrated that the fraction of CD4/CD8 cytotoxic T cells among total immune cells were significantly reduced at relapse compared to those at pretreatment or at BA remission. Furthermore, T cell exhaustion at relapse was demonstrated by upregulated immune checkpoint molecules TIGIT, CD96, and LAG3 in CD4/CD8 cytotoxic T cells. Ex vivo stimulation induced T cell activation to produce IFNγ and IL-2 in the samples collected at remission, but not at relapse. These data showed an acquisition of exhausted T cell state at relapse. In contrast, a significant elevation in myeloid cells was detected in samples at relapse compared to those at pretreatment or at BA remission. Myeloid-derived suppressive cells were enriched at relapse. Together, these data may indicate tumor immune escape at relapse by inducing a state of T cell exhaustion and expanded MDSCs. To further understand the tumor microenvironment, we performed longitudinal profiling of cytokines in patient plasma. A panel of soluble checkpoint molecules including PD-L1, PD-L2, TIM3, and LAG3 were significantly decreased at remission, compared to baseline and resurrected back to baseline at relapse. Moreover, soluble IL2 receptor (sIL2R) was significantly decreased at remission but was elevated at relapse at levels even higher than baseline. Ex vivo stimulation with IL-2 plus sIL2R enhanced cell growth compared to IL-2, or sIL2R alone in the sample collected at pretreatment, but diminished cell growth in samples collected at relapse. This suggests that sIL2R may play a suppressive function for T cell expansion and activation. Conclusion This study is the first study reporting on exploring potential mechanisms of resistance to BA therapy in patients with MCL. We demonstrate a reprogrammed tumor-immune landscape inducing T cell exhaustion, enrichment of suppressive MDSC, and elevated sIL-2R and check point molecules leading to resistance and relapses after BA therapy. Citation Format: Vivian Changying Jiang, Dapeng Hao, Preetesh Jain, Yijing Li, Qingsong Cai, Yang Liu, Yuxuan Che, Jovanny Vargas, Yixin Yao, Lei Nie, Enyu Dai, Guangchun Han, Ruiping Wang, Kunal Rai, Andrew Futreal, Christopher Flowers, Linghua Wang, Michael Wang. Multi-omics profiling can predict for relapse and response to brexucabtagene autoleucel CAR T-cell therapy in patients with mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 771.

High lake levels were developed in wide areas of western China during the late Quaternary as revealed by an increasing number of studies on dating shorelines; however, whether the high lake level of these lakes occurred simultaneously and what drove them are unresolved problems. In this study, in the Jilantai sub-depression within the Jilantai–Hetao Basin, dating results of the single-aliquot quartz and potassium-enriched feldspar (K-feldspar) single grains from the sediments at the western bank of the present Jilantai Salt Lake indicate that the ∼1070 m and 1060–1050 m shorelines formed after ∼136 ka and 49–22 ka, respectively, supporting the hypothesis that higher lake levels developed at intervals of marine isotope stage (MIS) 5e and MIS 3a–Last Glacial Maximum (LGM) in the Jilantai sub-depression. Considering the previous assumption that the lake at an altitude of 1080–1050 m was fed mainly by water from the Yellow River, the changes in the lakeshore landform features are sensitive to climate change in the Jilantai area, among which the development of lakeshore during MIS 3a–LGM was likely a geomorphic response to the development of dry climatic conditions rather than a representative of the humid climate in arid regions.

Four commonly found pesticides (alachlor, atrazine, metolachlor, and simazine) in surface water were determined using dispersive pipette extraction followed by gas chromatography–mass spectrometry....

Background Mantle Cell Lymphoma (MCL) is a rare incurable non-Hodgkin's lymphoma despite remarkable recent therapeutic innovations such as CAR T cell therapies. Drug sensitivity and immunotherapy efficacy assays using preclinical tumor models are important in new therapy development. As for preclinical tumor models, in addition to the commonly used mouse model such as patient-derived xenograft (PDX) and genetically engineered mouse models, a 3 rd tumor model, the patient-derived organoid (PDO) in 3D culture model has been established in many types of solid tumors. However, no PDO models have been generated from MCL yet. We have established MCL PDO model by optimizing cell isolation, culture add-ons, spatial and temporal conditions. Our protocol presents a versatile MCL PDO platform, suitable for quick drug screens and applicable for rapid immunotherapy evaluation. For proof of concept demonstration, we tested the therapeutic efficacy of CD19-targeted CAR T-cell in MCL-specific PDOs. The established PDO procedures can be used for diverse biopsies including whole blood, apheresis, bone marrow, lymph node and previously established PDX tumors. Methods For human blood, bone marrow and apheresis biospecimens, we first eliminated red blood cells using RBC lysis buffer. The cells were then spun down and cell pellets were resuspended in culture medium and aliquoted and spun-down into V-shaped 96-well plates at 1-6´10 6 cells per well. After overnight incubation, the cell culture medium was replaced by 60 ml of precooled 60% Matrigel per well. The 3D formed aggregates were gently transferred into 24-well plate and feed the solidified domes with the medium containing an in-house cytokine cocktail. The developed organoids reach the drug screening optimal phase within 3-6 days or it can be further expanded for future use. For PDX tumor and human lymph node biospecimens, we first sliced tumor tissue using surgical scalpel blade, which resulted in small tissue pieces that were then resuspended in HBSS. Debris (>100 mm) were removed by brief gravity sedimentation. The resultant tissue pieces were then resuspended in 60% Matrigel in culture medium and dispensed onto pre-warmed 24-well plates. The primary tumor organoids were observed for 3-5 days before drug testing on processed for further expansion. For organoid passaging, primary organoids were extracted in pre-cooled medium by mechanically breaking the gel domes. The extracted organoids were digested by collagenase/dispase. The resultant MCL and stromal cells were enforced to aggregate in V-shape wells as described in the apheresis procedure. Organoid cell composition was examined using FACS. Drug sensitivity and T-cell activity against the MCL organoids were assessed using CellTiter-Glo 3D kit. Results We have successfully established and optimized the PDO procedure from diverse MCL biopsies (Fig. 1A). The success rate of MCL organoid from apheresis was > 80% with even higher success rates when using PDX tumors, lymph node and bone marrow samples (> 90%). One critical step for processing the biopsies is preserving the original stromal cells. FACS analysis showed that although Conclusion We have established an MCL PDO platform which is time-efficient, labor-saving, cost-effective and highly reproducible. This platform provides a rapid approach for immune cell activity assays and drug screening. The organoids have been successfully used to generate PDX models. This platform can also be used for investigating the mechanism of drug resistance in the context of different TMEs. Figure 1 Figure 1. Disclosures Wang: Acerta Pharma: Consultancy, Honoraria, Research Funding; BioInvent: Research Funding; Pharmacyclics: Consultancy, Research Funding; Lilly: Research Funding; CStone: Consultancy; Oncternal: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; OMI: Honoraria; Newbridge Pharmaceuticals: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Physicians Education Resources (PER): Honoraria; Mumbai Hematology Group: Honoraria; InnoCare: Consultancy, Research Funding; Anticancer Association: Honoraria; VelosBio: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Juno: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria; Bayer Healthcare: Consultancy; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Imedex: Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Scripps: Honoraria; Dava Oncology: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Clinical Care Options: Honoraria; Chinese Medical Association: Honoraria; BGICS: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria.

Background Mantle cell lymphoma (MCL) is a rare and aggressive B-cell lymphoma characterized by poor prognosis. Although remarkable therapeutic advances have been made by covalent Bruton's tyrosine kinase (BTK) inhibition and CAR T cell therapy, therapeutic resistance inevitably occurs and leads to dismal clinical outcome. Pirtobrutinib (LOXO-305) is a next-generation, highly selective and non-covalent BTK inhibitor. A phase 1/2 BRUIN study showed that pirtobrutinib demonstrated promising efficacy in heavily pretreated MCL patients with or without prior covalent BTK inhibition. Here, we investigated the mechanism of action of pirtobrutinib in MCL cells in vitro and proposed the potential combination therapy in a venetoclax-resistant xenograft model. Methods MCL cell proliferation was monitored by trypan blue exclusion assay after 24-, 48- and 72-hour treatment with pirtobrutinib and ibrutinib. We performed Annexin V/PI staining to measure the apoptosis inductive effects. Cell cycle analysis using propidium iodide (PI) DNA staining was conducted to compare cell cycle progression kinetics between pirtobrutinib and ibrutinib. We performed RNAseq analysis in Z138 cells to compare differentially expressed genes (DEGs) between pirtobrutinib and ibrutinib treatment. Western blotting was utilized to detect specific signaling proteins. Mino-venetoclax-R cells were inoculated subcutaneously into NSG mice and used for in vivo drug efficacy determination. Results Compared to covalent BTK inhibitor ibrutinib, the novel non-covalent BTK inhibitor pirtobrutinib was more potent in inhibiting MCL cell proliferation in a panel of MCL cell lines, especially in ibrutinib/venetoclax resistant cell lines (pirtobrutinib vs. ibrutinib, p Conclusions Pirtobrutinib overcame both ibrutinib and venetoclax resistance in MCL cells in vitro and in vivo. G2/M checkpoints and E2F targets pathways were significantly enriched in both cases. Pirtobrutinib & venetoclax showed better in vivo efficacy in MCL models than combination of ibrutinib & venetoclax. Figure 1 Figure 1. Disclosures Wang: Genentech: Consultancy; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; CAHON: Honoraria; InnoCare: Consultancy, Research Funding; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Lilly: Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; OMI: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy; Chinese Medical Association: Honoraria; Dava Oncology: Honoraria; Celgene: Research Funding; Mumbai Hematology Group: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; Newbridge Pharmaceuticals: Honoraria; CStone: Consultancy; BGICS: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Scripps: Honoraria; Epizyme: Consultancy, Honoraria; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; VelosBio: Consultancy, Research Funding; Anticancer Association: Honoraria.

Introduction FDA approval for CD19 chimeric antigen receptor (CAR) T therapy with Brexucabtagene Autoleucel (BA) for relapsed mantle cell lymphoma (MCL) is a milestone advance. However, most patients relapsed after Bruton's tyrosine kinase (BTK) inhibitor ibrutinib therapy and CAR T therapy, making it urgent and essential to uncover the underlying mechanisms that govern resistance to ibrutinib and BA, as well as potential therapeutic targets. Method Two cohorts of primary patient samples were longitudinally collected at pre- and post-ibrutinib therapy (cohort 1, n = 12, samples = 28) or at pre- and post-BA therapy (cohort 2, n = 15, samples = 39) and subject to single-cell RNA sequencing (Figure 1A). Two healthy PBMC samples were included as normal controls. All 67 patient samples were divided into four major groups based on clinical outcomes: samples with fast response to ibrutinib (IBN-S), samples with slow response (IBN-Slow), samples with resistance (IBN-R), and samples with dual resistance to ibrutinib and BA (Dual). Data was integrated across cohorts and experimental batches. In-silico isolation of B cells was followed by differential gene expression analysis using a mixed model with random effect accounting for inter-patient variation. Gene Set Enrichment Analysis (GSEA) was performed to link clinical outcomes to dysregulated cancer hallmarks. Trajectory analysis further modeled the sequential changes resulting in different clinical outcomes. Copy number variation (CNV) analysis was performed to infer chromosomal aberrations. Results A high degree of transcriptomic heterogeneity was observed in tumor cells among patients within the same or across different clinical outcomes. Differential gene expression analysis revealed a set of outcome-specific genes across all patients. A list of 37 genes was consistently altered between Dual and IBN-R samples across both cohorts (FDR To capture the transitions of biological processes between different clinical outcomes, we performed trajectory analysis. The major trajectory stemmed from normal to IBN-S samples, then branched into IBN-Slow and IBN-R/Dual, which further branched into IBN-R and Dual (Figure 1C). A list of differentially expressed genes near the bifurcation point of trajectories (IBN-R/Dual) was identified (adjusted p-value < 2e-16) . To further understand the evolution associated with therapeutic resistance at the genomic level, we conducted the CNV analysis. We then quantified the genome instability score using chromosomal aberrations and observed a significant positive association with tumor aggressiveness (ANOVA test, p-value < 2e-16). Dual samples had the highest score, followed by IBN-R, IBN-Slow, and IBN-S (Figure 1D). Of note, chr22p gain was exclusive to the CAR T-resistant samples (4 out of 6), with specific aberrations in immunoglobulin lambda variable (IGLV) and constant (IGLC) genes, suggesting its association with BA resistance. Together with previously identified DEGs, targetable molecules are under active investigation for therapeutic development to overcome BA resistance. Conclusion In this study, we found co-enrichment of the OXPHOS and MYC pathways in each resistant cohort, suggesting their predominant role in ibrutinib resistance and BA resistance. Trajectory analysis detected genes differentially expressed at the branch point, which may represent novel early drivers of therapeutic resistance and disease progression. Acquired genome instability is positively correlated with tumor aggressiveness. The exclusive chr22p gain and immunoglobulin lambda alterations in Dual samples may suggest novel therapeutic targets to overcome the resistance for relapsed MCL patients. Collectively, our findings gained novel insight into the underlying mechanisms of resistance to ibrutinib and BA, as well as therapeutic vulnerabilities that can be targeted to overcome resistance. Figure 1 Figure 1. Disclosures Jain: Kite: Consultancy; Lilly: Membership on an entity's Board of Directors or advisory committees. Wang: Hebei Cancer Prevention Federation: Honoraria; Dava Oncology: Honoraria; Genentech: Consultancy; Bayer Healthcare: Consultancy; Clinical Care Options: Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Imedex: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; InnoCare: Consultancy, Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Mumbai Hematology Group: Honoraria; DTRM Biopharma (Cayman) Limited: Consultancy; Epizyme: Consultancy, Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; VelosBio: Consultancy, Research Funding; CStone: Consultancy; BGICS: Honoraria; OMI: Honoraria; Anticancer Association: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; CAHON: Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Chinese Medical Association: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Moffit Cancer Center: Honoraria; Newbridge Pharmaceuticals: Honoraria; Scripps: Honoraria; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; BioInvent: Research Funding; Celgene: Research Funding; Lilly: Research Funding; Molecular Templates: Research Funding; Physicians Education Resources (PER): Honoraria.