ALBERT

Description: Biochemistry DOI: 10.1021/bi400618q

Description: Journal of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.5b01363

Description: Background: Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphoma and is a therapeutic challenge. Phosphoinositide-3 kinase (PI3K) has been shown to be an alternative survival pathway in relapsed/refractory MCL. KA2237 (designed by Karus Therapeutics Ltd, Oxfordshire, United Kingdom) is a dual inhibitor of the class I beta and delta isoforms of the 110 kDa catalytic subunit of PI3K. By selectively targeting PI3K-beta and -delta isoforms and preventing their activation, KA2237 may decrease proliferation and induce cell death in susceptible tumor cells. Methods: We assessed the effects of KA2237 on the in vitro cell proliferation of both ibrutinib-sensitive (Mino, Jeko-1, and Rec-1) and primary ibrutinib-resistant (Z-138 and Maver-1) cell lines, and acquired ibrutinib-resistant MCL cell line, Jeko-R. We also tested the viability of patient-derived xenograft (PDX) tumor cells to KA2237. We compared the efficacy of KA2237 with two other commercial PI3K inhibitors, duvelisib (IPI-145, Selleck) and idelalisib (Cal-101, Selleck). Also, we paired these three inhibitors (KA2237, duvelisib and idelalisib) each with ibrutinib to evaluate the potential synergistic effects of these combinations. Lastly, we also tested in vivo efficacy of KA2237 and its combination with ibrutinib in PDX tumor cells. Results: KA2237 inhibited cell proliferation in both ibrutinib-sensitive and ibrutinib-resistant cell lines in a dose-dependent and time-dependent manner. For Mino and Jeko-1, the IC50 was 4.8 uM and 2.9 uM and for Z-138 and Maver-1 cell lines, the IC50 was 0.6 uM and 0.1 uM, respectively. KA2237 also decreased cell viability of ibrutinib-sensitive and ibrutinib-resistant MCL PDX tumor cells. However, KA2237 did not decrease the cell viability of normal human peripheral blood mono-nuclear cells. KA2237 arrested phase G0/G1 in Rec-1 and Jeko-R cell lines. We detected the expression of PI3K isoforms in MCL, finding higher expression of PI3K β and δ in MCL-resistant cell lines as compared with sensitive cell lines. We found that KA2237 induced MCL cell apoptosis in a time-dependent and dose-dependent manner. In comparison with duvelisib and idelalisib, KA2237 achieved greater inhibition of cell viability, cell apoptosis and cell cycle arrest. Furthermore, we found synergistic effects of KA2237 and ibrutinib combination in several MCL cell lines and in PDX models. In an ibrutinib-resistant PDX model, KA2237 treated mice reduced tumor burden significantly compared with vehicle control, and higher tumor growth inhibition was achieved as compared with ibrutinib. Conclusion: The novel PI3K inhibitor, KA2237 may be a potential candidate for MCL therapy, especially in the ibrutinib-resistant cases. Disclosures Wang: Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Research Funding; BeiGene: Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Celgene: Research Funding.

Description: Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses to frontline therapy. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) has achieved 68% of overall response rate in relapsed/refractory mantle cell lymphoma (MCL) patients. However, the vast majority of MCL patients experience disease progression, demonstrating that standard-of-care approaches are failing and that a means for targeting ibrutinib resistant MCL is clinically needed. Our hypothesis is that the ibrutinib-resistant MCL may rely on the mitochondrial oxidative phosphorylation (OXPHOS) pathway to produce energy for tumor growth. In this study, we investigated the effects of IACS-010759, a small molecule mitochondrial complex I inhibitor discovered in MD Anderson Cancer Center which can block the OXPHOS pathway, to overcome ibrutinib resistance in MCL in vitro and in a patient-derived xenograft (PDX) model. Methods: The OXPHOS metabolic pathways were investigated by RNASeq in a panel of ibrutinib-sensitive and -resistant MCL samples. Cell growth inhibition assays were tested after 72-hour treatment with IACS-010759 in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, and ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, by CellTiter-Glo luminescent cell viability assay (Promega). Furthermore, an IBN-resistant MCL PDX model was established and the therapeutic effects and tolerability of IACS-010759 were investigated in the primary MCL-bearing PDX model. Results: We have done RNA sequencing (RNASeq) in 7 primary ibrutinib-resistant and 16 ibrutinib-sensitive MCL patient samples, and analyzed the data using Gene Set Enrichment Analysis (GSEA) software. The results demonstrated that the OXPHOS pathway was activated in the primary ibrutinib-resistant MCL cells but not ibrutinib-sensitive MCL cells. Based on the RNASeq data, we selected an OXPHOS inhibitor IACS-010759 to investigate its effects on both primary ibrutinib-resistant and ibrutinib-sensitive MCL cells in vitroand in PDX mice. IACS-010759 significantly inhibited cell proliferation in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, but not in ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, during a 72-hour incubation. Furthermore, the primary ibrutinib-resistant MCL PDX mice were administrated with 10 mg/kg IACS-10759 by oral gavage, for 28 days using a 5 on/2 off dosing schedule. Our data showed that IACS-010759 completely eradicated tumor growth in ibrutinib-resistant MCL PDX mice (n=5, p=0.045). All mice tolerated the treatment dose and no toxicity was found during 28 days of IACS-010759 treatment. Conclusions: The OXPHOS inhibitor IACS-010759 overcomes ibrutinib resistance both in vitro and in the PDX mouse model. The investigation of its mechanism-of-action is ongoing. IACS-010759 could have the potential for clinical use in ibrutinib-resistant relapsed/refractory MCL patients. Disclosures Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Juno Therapeutics: Research Funding; Asana biosciences, Beigene, Celgene, Juno, Kite, Onyx, Pharmacyclics: Research Funding; Dava Oncology: Honoraria; BeiGene: Research Funding; Acerta: Consultancy, Research Funding.

Description: Background: Imprecise correlation of treatment response in established cell line and cells obtained from patients (i.e., patient-primary) limits the preclinical investigation of novel compounds. Similarly, the "xenograft" models wherein human cancer cell lines are transplanted into immunocompromised mice do not represent the full spectrum of cancers. However, Patient-derived xenograft (PDX) mouse models have been shown to recapitulate the diversity of growth, metastasis, and histopathology of the original tumor. Based on our previously established mantle cell lymphoma (MCL) PDX model, we developed other B-cell lymphoma PDXs recapitulating tumor pathological and clinical characteristics, progression and response to therapeutic agents, this will provide an indispensable model system towards personalized treatment for B-cell lymphoma. Methods: We developed 34 PDX models with an implanted fetal bone chip for several B-cell lymphomas including marginal zone lymphoma (MZL), follicular lymphoma (FL), Burkitt's lymphoma (BL), and diffuse large B-cell lymphoma (DLBCL), and MCL. We tested the in vitro efficacy of a panel of drugs among freshly isolated tumor cells from patients and tumor cells from the PDX models. We also generated a drug-resistant MCL PDX model, compared the effect of targeted drugs on the tumor burden in the drug-resistant model and identified potential therapeutic opportunity with drug combinations. We validated combination therapy in vivoand conducted next generation sequencing (NGS) using a 1,212 gene panel (OncoPlus®) on DNA from primary patient cells. Results: We collected clinical samples from 34 patients with several types of B-cell lymphomas including MCL (n=21), DLBCL (n=3), FL (n=2), BL (n=1), and MZL (n=2). Of the 34 patients, 18 (53%) were newly diagnosed and untreated clinically, 11 (32%) patients were relapsed after treatment with 1-3 chemotherapy or targeted therapy treatments, and 5 (15%) patients were treated with one-dose therapy before sample collection. All of the tumor cells, from both the patient and PDXs, showed the same drug response pattern. Consecutive ibrutinib administration to PDX mice from PT1 during G4 induced the development of an ibrutinib-resistant tumor. The cell viability of isolated PDX tumor cells treated with ibrutinib was not significantly different between G1 and G2 nor was it different between G5 and G6 (p〉0.05). In addition, histological features were consistent with patient tumor histology. Furthermore, whole exome sequencing revealed fidelity between the patient and PDX tumor cells as well as between subsequent generations of the PDX. [B1] We[Z2] engrafted a patients tumor cells into NSG-hu mice to create an MCL-bearing PDX mouse model (PT28-PDX). G2 PDX cells were isolated and treated with a panel of drugs. We found that G2 PDX cells were most sensitive to bortezomib (BTZ) (Velcade®). Growth inhibition of the G2 PDX cells was significantly higher with BTZ compared to ibrutinib (p=0.002) and cells were most sensitive to BTZ compared with other agents (p≤ 0.002). Based on this finding, the patient was treated with Velcade®, rituximab and dexamethasone and responded to treatment. However, the G3 PDX became resistant to BTZ. She was then treated with a 1 cycle of rituximab and cytarabine. Soon after, we initiated three-drug combination treatment with lenalidomide (Len), rituximab (RTX) and dexamethasone (DEX) in PDX G4. Len+RTX+DEX significantly prolonged mouse survival indicating that this could be an effective regimen for this patient after BTZ relapse. As guided by the PDX, PT28 underwent Len+RTX+DEX regimen and her peripheral lymphocytosis disappeared demonstrating response. Conclusions: The PDX model with implanted fetal bone chip is a valid experimental platform that recapitulates tumor characteristics of B-cell lymphomas. Leveraging the PDX platform to identify and select drugs that are likely to be efficacious for individual patients and subsequently administering the promising agents to those who relapse after an initial therapy is the next milestone. Disclosures Wang: BeiGene: Research Funding; Asana BioSciences: Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Background: Aberrant B-cell receptor signaling is an important contributor to lymphomagenesis in mantle cell lymphoma (MCL). Bruton's Tyrosine Kinase (BTK), a component of the BCR signaling axis, has been validated as a clinically relevant target, and BTK inhibitor ibrutinib received FDA approval for treatment of MCL in 2013. Growing concerns that single agent ibrutinib exerts off-target effects that interfere with other treatments such as rituximab-induced antibody-dependent cell cytotoxicity limit its utility in combination treatments. In this study, we assessed the in vitro and in vivo effects of BGB-3111in MCL models. Methods: We performed cell viability assays with BGB-3111 treated MCL cell lines to determine inhibition of cellular proliferation. The same assays were conducted on primary human MCL cells and patient-derived xenograft (PDX) tumor samples. Dose-dependent inhibition of BTK auto-phosphorylation and inhibition of downstream targets such as PLC-γ were determined by phospho-protein immunoblotting and immunoprecipitation. A reverse-phase protein assay (RPPA) was conducted on BGB-3111-treated Mino cells to evaluate changes in MCL oncogenic signaling. Induction of apoptosis in MCL cells treated with increasing doses of BGB-3111 was quantified using flow cytometry. For in vivo experiments, an ibrutinib-sensitive MCL PDX mouse model was treated with 50 mg/kg/day BGB-3111 and monitored for mean tumor burden and survival. Results: BGB-3111 potently inhibited cell viability in a panel of MCL cell lines, with an activity range of 1-10 uM, and induced apoptosis in a dose-dependent manner in several MCL cell lines.BGB-3111 treatment of MCL cells demonstrated a dose-dependent decrease in p-BTK (Y223) and inhibition of downstream effectors without impacting total protein levels, while RPPA revealed upregulation of the PI3K-Akt signaling axes. In addition, BGB-3111 treatment did not impact phosphorylation of off-target kinases affected by ibrutinib treatment. In vivo, BGB-3111 suppressed tumor growth and prolonged tumor survival in BGB-3111 treated mice. Conclusion: The second generation BTK inhibitor BGB-3111 demonstrates selectivity for BTK in vitro and BTK inhibition in vivo. BGB-3111-treated PDX mouse models examining survival, tumor growth, and other factors point to BGB-3111 as an effective single agent BGB-3111 is being investigated in Phase I clinical trials. Disclosures Wang: Beigene: Employment. Wang:Asana BioSciences: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Honoraria; Acerta: Consultancy, Research Funding; Kite Pharma: Research Funding; BeiGene: Research Funding; Asana biosciences, Beigene, Celgene, Juno, Kite, Onyx, Pharmacyclics: Research Funding; Juno Therapeutics: Research Funding.

Description: Introduction Mantle cell lymphoma (MCL) is an aggressive lymphoma with elevated B-cell receptor activity. Ibrutinib (IBN), a Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have a response rate of 68% in relapsed or refractory MCL patients. However, with the emergence of IBN-resistant lymphomas, new therapies are needed. It is suspected that upregulation of the PI3K-Akt-mTOR pathway allows survival in the presence of IBN. CUDC-907 is a next generation PI3K and HDAC dual-inhibitor currently in phase II clinical trials. Our objective is to investigate the effects of CUDC-907 in IBN-resistant MCL cells in vitro and in PDX model. Methods MCL cells were seeded at 10,000 cells per well in 96-well plate and were treated with various doses of compounds at the following concentrations: CUDC-907/Ibrutinib 0.015, 0.05, 0.15, 0.5, 1.5, 5, and 15 uM. Cell viability was tested by CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation. Next, MCL cells were incubated with IBN at varying doses (0.39 uM, 1.56 uM, 6.25 uM), CUDC-907 (0.39 uM, 1.56 uM, 6.25 uM), and IBN+CUDC-907 (0.39 uM, 1.56 M, 6.25 uM) for 24 hours. Apoptosis was detected by Annexin V-binding assay. In patient derived xenograft (PDX) model: CUDC-907 was administered at a dose of 50 mg/kg in ibrutinib-resistant MCL-bearing PDX mice daily. Tumor volumes were measured as the length X width2 X 0.5 weekly. Toxicity was also observed every week. Mice were sacrificed once diameter of tumor mass reached 15 mm size. Results CUDC-907 inhibited the growth of both ibrutinib-sensitive and resistant MCL cells in vitro. Sensitive cell lines include: Rec-1, Mino, and JVM-13 with IC50 values of 1.1 nM, 1.0 nM, and 5 nM respectively. Resistant cell lines include: Granta-519, Maver-1, and Z-138 with IC50 values of 2 nM, 3 nM, and 1.5 nM respectively. All tested cell lines were more sensitive to CUDC-907 than to ibrutinib. In addition, combination treatments of CUDC-907 and IBN increased cell death in comparison to single agent treatments. Next, one pair of MCL cell lines, Jeko-1 (sensitive to IBN) and Jeko-R (resistant to IBN) were treated for 24 hours with varying doses of CUDC-907 or ibrutinib either as single agent inhibitors or in combination therapies. The results demonstrated that CUDC-907 induced apoptosis in both Jeko-1 and Jeko-R cell lines in a dose-dependent manner. Combination therapies increased cell death in a dose-dependent manner as well. In PDX model, tumor volume in treated mice of ibrutinib-resistant PDX decreased significantly compared with vehicle control (pvalue = 0.032). Control mice also weighed considerably more than treated mice (p value = 0.073). Common toxicities included a decrease in body mass for first 28 days of treatment. Conclusion CUDC-907, a dual inhibitor of PI3K-Akt-mTOR and HDAC, inhibits tumor growth of ibrutinib-resistant MCL in vitro and in PDX model. It would be a potential drug for the patients with ibrutinib-resistant/relapsed MCL. Disclosures Wang: Celgene: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Research Funding; Juno Therapeutics: Research Funding; Asana BioSciences: Research Funding.

Description: Background Mantle cell lymphoma (MCL) is a B-cell malignancy with a broad spectrum of clinical, pathological, and biological features, and clinical evolution is usually very aggressive with short responses to treatment and frequent relapses. Ibrutinib is considered the drug of choice in relapsed-refractory cases but patients may develop resistance as some patients in complete remission (CR) have not relapsed yet and are in long term follow-up. The understanding of the resistance mechanisms and the emergence of new drugs targeting key oncogenic mechanisms are providing the basis for designing innovative therapeutic strategies to overcome ibrutinib resistance, both in preclinical studies and preliminary clinical trials. Methods We identified differentially expressed genes (DEGs) in 7 ibrutinib-primary resistant MCL patient samples compared with 16 ibrutinib-sensitive MCL patient samples by next generation sequencing (RNA-Seq) and top28-gene signature were developed via Gene Set Enrichment Analysis (GSEA) analysis of RNA-Seq data, and we also verified the expression level of these genes in ibrutinib-resistant and-sensitive MCL patient samples using Real time-PCR, and then a secondary focus of this study was to identify potential predictive biomarkers for therapy in relapsed or refractory MCL. In order to place the gene expression data into a biological context, Ingenuity Pathway Analysis (IPA) software was used to assign the DEGs to know the canonical pathways and functional networks in order to predict the biological functions of the transcriptional changes. Results We identified top-28 DEGs in five ibrutinib-resistant MCL patient samples compared with four ibrutinib-sensitive MCL patient samples. We performed gene enrichment and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG pathway) of differentially expressed genes of each samples and verified the predicted genes using Real time-PCR, which were truly related to MCL and not false positive results. Moreover, Using IPA software, we identified that the enriched biological functions in the MCL ibrutinib-resistant patient samples were Oxidative phosphorylation, Mitochondrial Dysfunction and TCA Cycle (Eukaryotic), which were enriched biological functions in the analysis of RNA-Seq data, and which may be targeted by oncogenic events in MCL, and they may influence the tumor response to new therapeutic agents. We also found that expression of these genes (SEPT3, FASN, IDH3A, SLC1A5, INPP5J, CCT5, MTHFD1) was significantly increased in five ibrutinib-resistant MCL patient samples compared with four ibrutinib-sensitive MCL patient samples, and we used IPA to identify some functionally related genes with these genes increased in MCL ibrutinib-resistant patient samples and built networks based on the molecular relationships most relevant to this project. Conclusion These data identify a genomic basis for ibrutinib-primary resistance in MCL and provide the important insights into the strategy to address the problem of ibrutinib-resistance, and will hopefully allow more tailored and specific therapies to be designed. Disclosures Wang: Onyx: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Kite Pharma: Research Funding; Asana BioSciences: Research Funding; BeiGene: Research Funding; Juno Therapeutics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.