Ofatumumab plus HyperCVAD/HD-MA induction leads to high rates of minimal residual disease negativity in patients with newly diagnosed mantle cell lymphoma: Results of a phase 2 study.

BACKGROUND: Ofatumumab is a humanized type 1 anti-CD20 monoclonal antibody. Preclinical studies show improved complement-mediated cytotoxicity (CMC) compared to rituximab in mantle cell lymphoma (MCL). This study evaluates the safety and efficacy of combining ofatumumab with HyperCVAD/MA (O-HyperCVAD) in newly diagnosed MCL. METHODS: In this single-arm phase 2 study, 37 patients were treated with the combination of O-HyperCVAD for 4 or 6 cycles, followed by high dose chemotherapy and autologous stem cell transplant. Primary objectives were overall response rate (ORR) and complete response (CR) rate at the end of therapy. Secondary objectives included minimal residual disease (MRD) negativity, progression-free survival (PFS), and overall survival (OS). RESULTS: Median age was 60 years; ORR was 86% and 73% achieved a CR by modified Cheson criteria. The MRD negativity rate was 78% after 2 cycles of therapy, increasing to 96% at the end of induction; median PFS and OS were 45.5 months and 56 months, respectively. Achieving a post-induction CR by both imaging and flow cytometry was associated with improved PFS and OS. Early MRD negativity (post-2 cycles) was also associated with an improved PFS but not OS. There were 3 deaths while on therapy, and grades 3 and 4 adverse events (AEs) were observed in 22% and 68% of the patients. CONCLUSION: The addition of ofatumumab to HyperCVAD/HD-MA led to high rates of MRD negativity by flow cytometry in patients with newly diagnosed MCL. Achieving a CR post-induction by both imaging and flow cytometry is associated with improved overall survival.

Pevonedistat, a NEDD8-activating enzyme inhibitor, is active in mantle cell lymphoma and enhances rituximab activity in vivo.

Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 µM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-κB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-κB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL.

Entinostat, a novel histone deacetylase inhibitor is active in B-cell lymphoma and enhances the anti-tumour activity of rituximab and chemotherapy agents.

Histone deacetylases (HDACs) inhibitors are active in T-cell lymphoma and are undergoing pre-clinical and clinical testing in other neoplasms. Entinostat is an orally bioavailable class I HDAC inhibitor with a long half-life, which is under evaluation in haematological and solid tumour malignancies. To define the activity and biological effects of entinostat in B-cell lymphoma we studied its anti-tumour activity in several rituximab-sensitive or -resistant pre-clinical models. We demonstrated that entinostat is active in rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL) and primary tumour cells isolated from lymphoma patients (n = 36). Entinostat exposure decreased Bcl-XL (BCL2L1) levels and induced apoptosis in cells. In RSCL and RRCL, entinostat induced p21 (CDKN1A) expression leading to G1 cell cycle arrest and exhibited additive effects when combined with bortezomib or cytarabine. Caspase inhibition diminished entinostat activity in some primary tumour cells suggesting that entinostat has dual mechanisms-of-action. In addition, entinostat increased the expression of CD20 and adhesion molecules. Perhaps related to these effects, we observed a synergistic activity between entinostat and rituximab in a lymphoma-bearing severe combined immunodeficiency (SCID) mouse model. Our data suggests that entinostat is an active HDAC inhibitor that potentiates rituximab activity in vivo and supports its further clinical development in B-cell lymphoma.

Downregulation of deoxycytidine kinase in cytarabine-resistant mantle cell lymphoma cells confers cross-resistance to nucleoside analogs gemcitabine, fludarabine and cladribine, but not to other classes of anti-lymphoma agents.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. Implementation of high-dose cytarabine (araC) into induction therapy became standard-of-care for all newly diagnosed younger MCL patients. However, many patients relapse even after araC-based regimen. Molecular mechanisms responsible for araC resistance in MCL are unknown and optimal treatment strategy for relapsed/refractory MCL patients remains elusive. METHODS: Five araC-resistant (R) clones were derived by long-term culture of five MCL cell lines (CTRL) with increasing doses of araC up to 50 microM. Illumina BeadChip and 2-DE proteomic analysis were used to identify gene and protein expression changes associated with araC resistance in MCL. In vitro cytotoxicity assays and experimental therapy of MCL xenografts in immunodeficient mice were used to analyze their relative responsiveness to a set of clinically used anti-MCL drugs. Primary MCL samples were obtained from patients at diagnosis and after failure of araC-based therapies. RESULTS: Marked downregulation of deoxycytidine-kinase (DCK) mRNA and protein expression was identified as the single most important molecular event associated with araC-resistance in all tested MCL cell lines and in 50% primary MCL samples. All R clones were highly (20-1000x) cross-resistant to all tested nucleoside analogs including gemcitabine, fludarabine and cladribine. In vitro sensitivity of R clones to other classes of clinically used anti-MCL agents including genotoxic drugs (cisplatin, doxorubicin, bendamustine) and targeted agents (bortezomib, temsirolimus, rituximab) remained unaffected, or was even increased (ibrutinib). Experimental therapy of immunodeficient mice confirmed the anticipated loss of anti-tumor activity (as determined by overall survival) of the nucleoside analogs gemcitabine and fludarabine in mice transplanted with R clone compared to mice transplanted with CTRL cells, while the anti-tumor activity of cisplatin, temsirolimus, bortezomib, bendamustine, cyclophosphamide and rituximab remained comparable between the two cohorts. CONCLUSIONS: Acquired resistance of MCL cells to araC is associated with downregulation of DCK, enzyme of the nucleotide salvage pathway responsible for the first phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. The data suggest that nucleoside analogs should not be used in the therapy of MCL patients, who relapse after failure of araC-based therapies.

The novel proteasome inhibitor carfilzomib induces cell cycle arrest, apoptosis and potentiates the anti-tumour activity of chemotherapy in rituximab-resistant lymphoma.

Targeting the proteasome system with bortezomib (BTZ) results in anti-tumour activity and potentiates the effects of chemotherapy/biological agents in multiple myeloma and B-cell lymphoma. Carfilzomib (CFZ) is a more selective proteasome inhibitor that is structurally distinct from BTZ. In an attempt to characterize its biological activity, we evaluated CFZ in several lymphoma pre-clinical models. Rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL), and primary tumour cells derived from B-cell lymphoma patients were exposed to CFZ or BTZ. Cell viability and changes in cell cycle were determined. Western blots were performed to detect PARP-cleavage and/or changes in Bcl-2 (BCL2) family members. CFZ was 10 times more active than BTZ and exhibited dose- and time-dependent cytotoxicity. CFZ exposure induced apoptosis by upregulation of Bak (BAK1) and subsequent PARP cleavage in RSCL and RRCL; it was also partially caspase-dependent. CFZ induced G2/M phase cell cycle arrest in RSCL. CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti-tumour activity of chemotherapy agents. Our data suggest that CFZ is able to overcome resistance to chemotherapeutic agents, upregulate pro-apoptotic proteins to promote apoptosis, and induce G2/M cell cycle arrest in lymphoma cells. Our pre-clinical data supports future clinical evaluation of CFZ in B-cell lymphoma.

MLN2238, a proteasome inhibitor, induces caspase-dependent cell death, cell cycle arrest, and potentiates the cytotoxic activity of chemotherapy agents in rituximab-chemotherapy-sensitive or rituximab-chemotherapy-resistant B-cell lymphoma preclinical models.

To further develop therapeutic strategies targeting the proteasome system, we studied the antitumor activity and mechanisms of action of MLN2238, a reversible proteasome inhibitor, in preclinical lymphoma models. Experiments were conducted in rituximab-chemotherapy-sensitive cell lines, rituximab-chemotherapy-resistant cell lines (RRCL), and primary B-cell lymphoma cells. Cells were exposed to MLN2238 or caspase-dependent inhibitors, and differences in cell viability, alterations in apoptotic protein levels, effects on cell cycle, and the possibility of synergy when combined with chemotherapeutic agents were evaluated. MLN2238 showed more potent dose-dependent and time-dependent cytotoxicity and inhibition of cell proliferation in lymphoma cells than bortezomib. Our data suggest that MLN2238 can induce caspase-independent cell death in RRCL. MLN2238 (and to a much lesser degree bortezomib) reduced RRCL S phase and induced cell cycle arrest in the G2/M phase. Exposure of rituximab-chemotherapy-sensitive cell lines and RRCL to MLN2238 potentiated the cytotoxic effects of gemcitabine, doxorubicin, and paclitaxel and overcame resistance to chemotherapy in RRCL. MLN2238 is a potent proteasome inhibitor active in rituximab-chemotherapy-sensitive and rituximab-chemotherapy-resistant cell models and potentiates the antitumor activity of chemotherapy agents and has the potential of becoming an effective therapeutic agent in the treatment of therapy-resistant B-cell lymphoma.

AMG176, an MCL-1 inhibitor, is active in pre-clinical models of aggressive B-cell lymphomas.

Upregulation of the anti-apoptotic protein MCL-1 has been implicated in chemotherapy resistance and poor clinical outcomes in B-cell lymphoma (BCL). We report the activity of AMG176, a direct, selective MCL-1 inhibitor, in preclinical models of BCL. A panel of cell lines representing diffuse large B-cell lymphoma (DLBCL), double-hit lymphoma (DHL) and Burkitt's lymphoma (BL) was selected. AMG176 induced apoptotic cell death in a dose- and time-dependent manner in all BCL cell lines. Baseline MCL-1 expression was not predictive of response. AMG176 exhibited impressive synergy with venetoclax and chemotherapeutic agents, less so with proteasomal inhibitors, and antagonism with anti-CD20 monoclonal antibodies. The activity of AMG176 could not be confirmed in murine models of BCL. Combination therapy targeting MCL-1 and BCL-2 may provide an alternative therapeutic approach in BCL, however optimal patient selection will remain the key to obtaining high response rates and tolerability.

Population Pharmacokinetics and Pharmacodynamics of Carfilzomib in Combination with Rituximab, Ifosfamide, Carboplatin, and Etoposide in Adult Patients with Relapsed/Refractory Diffuse Large B Cell Lymphoma.

BACKGROUND: In patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), salvage chemotherapy regimens (e.g., rituximab, ifosfamide, carboplatin, and etoposide, R-ICE) yield poor outcomes. Carfilzomib, an irreversible proteasome inhibitor, can overcome acquired rituximab-chemotherapy resistance and, when combined with R-ICE, improves outcomes in patients with R/R DLBCL. OBJECTIVE: This analysis aimed to develop a population pharmacokinetic/pharmacodynamic (PK/PD) model for carfilzomib in R/R DLBCL patients. PATIENTS AND METHODS: In a single-center, open-label, prospective phase 1 study, patients received carfilzomib (10, 15, or 20 mg/m2) on days 1, 2, 8, and 9, and standard doses of R-ICE on days 3-6 every 21 days (maximum of three cycles). Carfilzomib plasma concentrations up to 24 h postinfusion were measured by liquid chromatography coupled with tandem mass spectrometry. Proteasome activity (PD biomarker) in peripheral blood mononuclear cells was assessed on days 1-2 with sparse sampling. PK/PD models were developed using NONMEM v7.4.1 interfaced with Finch Studio v1.1.0 and PsN v4.7.0. Model selection was guided by objective function value, goodness-of-fit, and visual predictive checks. Stepwise covariate modeling was used for covariate selection. RESULTS: Twenty-eight patients were enrolled in the PK/PD analysis, from whom 217 PK samples and 127 PD samples were included. Carfilzomib PK was best described by a two-compartment model with linear disposition (typical total clearance of 133 L/h). Proteasome activity was best characterized using a turnover model with irreversible inactivation. All parameters were estimated with good precision. No statistically significant covariates were identified. CONCLUSIONS: A validated population-based PK/PD model of carfilzomib was developed successfully. Further research is needed to identify sources of variability in response to treatment with carfilzomib in combination with R-ICE. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier number NCT01959698.

Early changes in soluble intracellular adhesion molecule-1 as prognostic biomarkers to immune checkpoint inhibitor.

Serologic biomarker to predict clinical outcome is needed for immune checkpoint inhibitors (ICIs). We evaluated soluble intercellular adhesion molecules-1 (sICAM-1) as a predictor of response to ICIs treatment. Ninety-five patients with cancer treated with ICI were studied. The serum sICAM-1 levels of baseline, post two cycle therapy and end of therapy (EOT) were measured by enzyme-linked immunoassay. We randomly assigned the patients into the primary cohort (n = 47) and validation cohort (n = 48). Serum sICAM-1 post two cycle (277.7 ± 181.6 ng/mL) and EOT (403.9 ± 218.9 ng/mL) were significantly elevated compared to baseline (244.8 ± 153.8 ng/mL, p = 0.008 and p = 0.004, respectively). Early changes of sICAM-1 (ΔsICAM-1), deemed as sICAM-1 after two cycles minus baseline, were assessed. Following ICI treatments, responders had significantly lower ΔsICAM-1 compared with nonresponders in the primary cohort (p = 0.040) and the validation cohort (p = 0.026). High ΔsICAM-1 was strongly associated with inferior progression-free survival (PFS; (primary cohort: p = 0.001 and validation cohort: p = 0.002) and overall survival (OS; (primary cohort: p < 0.001 and validation cohort: p = 0.007). The ΔsICAM-1 remained independently associated with worse PFS and OS in the primary cohort and the validation cohort. Subgroup analysis indicated patients whose sICAM-1 significantly elevated had shorter PFS and OS in both anti-PD-1 and anti-PD-L1 treatment groups. Early change of serum sICAM-1 could be used to monitor and predict clinical benefit of ICI therapy in patients with solid cancer.

Carfilzomib combined with rituximab, ifosfamide, carboplatin, and etoposide for relapsed or refractory DLBCL.

The CORAL study highlighted the need to develop novel salvage regimens in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) previously treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. Carfilzomib (CFZ) can overcome rituximab chemotherapy resistance in lymphoma preclinical models by targeting the ubiquitin-proteasome system. We conducted an investigator initiated, single-center, open-label, prospective phase 1 study evaluating the safety and efficacy of CFZ in combination with rituximab, ifosfamide, carboplatin, and etoposide (C-R-ICE) in high-dose chemotherapy with autologous stem cell transplant (HDC-ASCT) eligible patients with R/R DLBCL (NCT01959698). In the dose-escalation phase, 18 patients were enrolled at 6 dose levels with no dose-limiting toxicities noted. CFZ 45 mg/m2 was selected as the recommended dose for expansion. Eleven additional patients were enrolled in the dose-expansion phase. Overall response rate (ORR) was 66% (48% CR; 17% PR); 52% patients underwent HDC-ASCT. An ORR of 85% was observed in patients with nongerminal center B-cell-like (non-GCB) DLBCL compared with only 13% in those with GCB DLBCL. Median progression-free survival (PFS) was 15.2 months (5.1 months, not reached [NR]), and median overall survival (OS) was 22.6 months (6.8 months, NR). Patients with non-GCB subtype had a significantly longer PFS (NR vs 6.6 months; P = .0001) and OS (NR vs 6.6 months; P = .001) than those with GCB subtype. C-R-ICE is well tolerated in patients with R/R DLBCL with toxicities comparable to rituximab, ifosfamide, carboplatin, and etoposide therapy. Our data show that patients with non-GCB DLBCL benefit significantly from incorporating CFZ into second-line therapy and HDC-ASCT.

Ofatumumab demonstrates activity against rituximab-sensitive and -resistant cell lines, lymphoma xenografts and primary tumour cells from patients with B-cell lymphoma.

Ofatumumab is a new monoclonal antibody (mAb) targeting a novel membrane-proximal epitope on CD20. To better define ofatumumab's activity, we conducted pre-clinical studies in rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL), ofatumumab-exposed cell lines (OECLs), primary lymphoma cells, and a lymphoma xenograft model. RRCL and OECL were generated by repeated exposure of sensitive cells to escalating doses of rituximab or ofatumumab ± human serum. Antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) assays were performed to assess cellular sensitivity to rituximab or ofatumumab. Ofatumumab elicited a higher rate of CMC in RSCL, RRCL and primary tumour cells. The chronic exposure of lymphoma cells to ofatumumab resulted in rituximab resistance but less ofatumumab resistance. In an in vivo severe combined immunodeficiency mouse model of human lymphoma, ofatumumab prolonged median survival compared to rituximab. While rituximab CMC diminished with CD20 down-regulation in RRCL passages, ofatumumab activity in vitro diminished to a lesser degree. Our data suggest that ofatumumab is more potent than rituximab in rituximab-sensitive or rituximab-resistant models and has the potential to decrease the development of biological resistance in patients with repeated exposure to anti-CD20 mAbs.

Distinct molecular mechanisms responsible for bortezomib-induced death of therapy-resistant versus -sensitive B-NHL cells.

Resistance to currently available therapies is a major impediment to the successful treatment of hematological malignancies. Here, we used a model of therapy-resistant B-cell non Hodgkin lymphoma (B-NHL) developed in our laboratory along with primary B-NHL cells to study basic mechanisms of bortezomib activity. In resistant cells and a subset of primary B-NHLs, bortezomib treatment led to stabilization of Bak and subsequent Bak-dependent activation of apoptosis. In contrast to sensitive cells that die strictly by apoptosis, bortezomib was capable of killing resistant cells through activation of apoptosis or caspase-independent mechanism(s) when caspases were pharmacologically inhibited. Our data demonstrate that bortezomib is capable of killing B-NHL cells via multiple mechanisms, regardless of their basal apoptotic potential, and contributes to growing evidence that proteasome inhibitors can act via modulation of B-cell lymphoma 2 (Bcl-2) family proteins. The capacity of bortezomib to act independently of the intrinsic apoptotic threshold of a given B-NHL cell suggests that bortezomib-based therapies could potentially overcome resistance and result in relevant clinical activity in a relapsed/refractory setting.

Small molecule MMRi62 targets MDM4 for degradation and induces leukemic cell apoptosis regardless of p53 status.

MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interact via their RING domains and form a heterodimer polyubiquitin E3 ligase essential for p53 degradation. MDM4 also forms heterodimer E3 ligases with MDM2 isoforms that lack p53-binding domains, which regulate p53 and MDM4 stability. We are working to identify small-molecule inhibitors targeting the RING domain of MDM2-MDM4 (MMRi) that can inactivate the total oncogenic activity of MDM2-MDM4 heterodimers. Here, we describe the identification and characterization of MMRi62 as an MDM4-degrader and apoptosis inducer in leukemia cells. Biochemically, in our experiments, MMRi62 bound to preformed RING domain heterodimers altered the substrate preference toward MDM4 ubiquitination and promoted MDM2-dependent MDM4 degradation in cells. This MDM4-degrader activity of MMRi62 was found to be associated with potent apoptosis induction in leukemia cells. Interestingly, MMRi62 effectively induced apoptosis in p53 mutant, multidrug-resistant leukemia cells and patient samples in addition to p53 wild-type cells. In contrast, MMRi67 as a RING heterodimer disruptor and an enzymatic inhibitor of the MDM2-MDM4 E3 complex lacked MDM4-degrader activity and failed to induce apoptosis in these cells. In summary, this study identifies MMRi62 as a novel MDM2-MDM4-targeting agent and suggests that small molecules capable of promoting MDM4 degradation may be a viable new approach to killing leukemia cells bearing non-functional p53 by apoptosis.

Distinct cellular and therapeutic effects of obatoclax in rituximab-sensitive and -resistant lymphomas.

Bcl-2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3-mimetic, has been designed to specifically target and counteract anti-apoptotic Bcl-2 proteins. We evaluated the biological effects of obatoclax on the anti-tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy-sensitive (RSCL), -resistant cell lines (RRCL) and primary tumour-cells derived from patients with B-cell lymphomas (N=39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase-independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient-derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin-1 knockdown. In summary, obatoclax is an active Bcl-2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response.

Pevonedistat, a NEDD8-Activating Enzyme Inhibitor, Induces Apoptosis and Augments Efficacy of Chemotherapy and Small Molecule Inhibitors in Pre-clinical Models of Diffuse Large B-cell Lymphoma.

We studied the biological activity of pevonedistat, a first-in-class NEDD8-activating enzyme (NAE) inhibitor, in combination with various cytotoxic chemotherapy agents and small molecule inhibitors in lymphoma pre-clinical models. Pevonedistat induced cell death in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) cell lines and to a lesser degree in germinal center B-cell (GCB) DLBCL cell lines. In pevonedistat sensitive cells, we observed inhibition of NFκB activity by p65 co-localization studies, decreased expression of BCL-2/BCL-XL and upregulation of BAK levels. Pevonedistat enhanced the activity of cytarabine, cisplatin, doxorubicin and etoposide in ABC-, but not in the GCB-DLBCL cell lines. It also exhibited synergy with ibrutinib, selinexor, venetoclax and A-1331852 (a novel BCL-XL inhibitor). In vivo, the combination of pevonedistat and ibrutinib or pevonedistat and cytarabine prolonged survival in SCID mice xenograft models when compared with monotherapy controls. Our data suggest that targeting the neddylation pathway in DLBCL is a viable therapeutic strategy and support further clinical studies of pevonedistat as a single agent or in combination with chemotherapy or novel targeted agents.

Metformin sensitizes therapeutic agents and improves outcome in pre-clinical and clinical diffuse large B-cell lymphoma.

BACKGROUND: The treatment of diffuse large B-cell lymphoma (DLBCL) is limited by the development of resistance to therapy, and there is a need to develop novel therapeutic strategies for relapsed and refractory aggressive lymphoma. Metformin is an oral agent for type 2 diabetes that has been shown to decrease cancer risk and lower mortality in other types of cancer. METHODS: We performed a retrospective analysis of the RPCCC database looking at patients with DLBCL treated with front-line chemotherapy. We also performed pre-clinical studies looking at the effect of metformin on cell viability, cell number, Ki67, ATP production, apoptosis, ROS production, mitochondrial membrane potential, cell cycle, effect with chemotherapeutic agents, and rituximab. Finally, we studied mouse models to see the anti-tumor effect of metformin. RESULTS: Among diabetic patients, metformin use was associated with improved progression-free survival (PFS) and overall survival (OS) compared to diabetic patients not on metformin. Our pre-clinical studies showed metformin is itself capable of anti-tumor effects and causes cell cycle arrest in the G1 phase. Metformin induces apoptosis, ROS production, and increased mitochondrial membrane permeability. Metformin exhibited additive/synergistic effects when combined with traditional chemotherapy or rituximab in vitro. In vivo, metformin in combination with rituximab showed improved survival compared with rituximab monotherapy. CONCLUSIONS: Our retrospective analysis showed that metformin with front-line chemotherapy in diabetic patients resulted in improved PFS and OS. Our pre-clinical studies demonstrate metformin has potential to re-sensitize resistant lymphoma to the chemo-immunotherapy and allow us to develop a hypothesis as to its activity in DLBCL.

Overcoming resistance to rituximab in relapsed non-Hodgkin lymphomas by antibody-polymer drug conjugates actively targeted by anti-CD38 daratumumab.

B-cell non-Hodgkin lymphomas (B-NHL) represent the most common type of hematologic malignancies in the Western hemisphere. The therapy of all B-NHL is based on the combination of different genotoxic cytostatics and anti-CD20 monoclonal antibody (mAb) rituximab. Unfortunately, many patients relapse after the mentioned front-line treatment approaches. The therapy of patients with relapsed/refractory (R/R) B-NHL represents an unmet medical need. We designed, developed and tested novel actively targeted hybrid mAb-polymer-drug conjugate (APDC) containing anti-CD20, anti-CD38 or anti-CD19 mAbs. Biocompatible copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) with cytostatic agent doxorubicin attached via stimuli-sensitive hydrazone bond were employed for the mAb grafting. Anti-lymphoma efficacy of the APDC nanotherapeutics was evaluated in vivo on a panel of three patient-derived lymphoma xenografts derived from two patients with R/R B-NHL and one patient with so far untreated B-NHL. In both PDX models derived from patients with R/R B-NHL, the targeting with anti-CD38 antibody daratumumab demonstrated highly improved anti-lymphoma efficacy compared to the targeting with anti-CD20 rituximab, two experimental anti-CD19 antibodies and non-targeted controls. The results represent a proof-of-concept of a new algorithm of personalized anti-tumor therapy based on highly innovative APDC biomaterials.

The adhesion molecule ICAM-1 in diffuse large B-cell lymphoma post-rituximab era: relationship with prognostic importance and rituximab resistance.

Intercellular adhesion molecule-1 (ICAM-1) is a cell-surface receptor contributing to lymphocyte homing, adhesion and activation. The prognostic significance of the protein is unknown in diffuse large B-cell lymphoma (DLBCL) in post-rituximab era. We detected expression of ICAM-1 immunohistochemically in 102 DLBCL tissue samples. Overexpression of ICAM-1 was found in 28 (27.5%) cases. In patients with low ICAM-1 expression levels, the addition of rituximab to CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy resulted in an improved overall response rate, progression-free survival (PFS) and overall survival (OS) (P=0.019, 0.01, 0.02). In pre-clinical models, we found that chronic exposure of cell lines to rituximab led to downregulation of ICAM-1 and acquirement of a rituximab resistant phenotype. In vitro exposure of rituximab resulted in rapid aggregation of B-cells regardless of the ICAM-1 expression levels. MTT assay showed knockdown of ICAM-1 could cause rituximab resistance. Neutralization of ICAM-1 did not affect rituximab activity in vitro and in vivo. Our data illustrated that in post-rituximab era, R-CHOP significantly improved the ORR, PFS and OS in ICAM-1 negative subset patients. Downregulation of ICAM-1 may contribute to rituximab resistance, and that rituximab, by promoting cell-cell aggregation, may sensitize cells to the cytotoxic effects of chemotherapy agents.

Expression level and DNA methylation status of glutathione-S-transferase genes in normal murine prostate and TRAMP tumors.

BACKGROUND: Glutathione-S-transferase (Gst) genes are downregulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors. METHODS: Primary and metastatic tumors were obtained from TRAMP mice, and normal prostates were obtained from strain-matched WT mice (n = 15/group). Quantitative real-time RT-PCR was used to measure GstA4, GstK1, GstM1, GstO1, and GstP1 mRNA expression, and Western blotting and immunohistochemical staining was used to measure GstM1 and GstP1 protein expression. MassARRAY Quantitative Methylation Analysis was used to measure DNA methylation of the 5' CpG islands of GstA4, GstK1, GstM1, GstO1, and GstP1. TRAMP-C2 cells were treated with the epigenetic remodeling drugs decitabine and trichostatin A (TSA) alone and in combination, and Gst gene expression was measured. RESULTS: Of the genes analyzed, GstM1 and GstP1 were expressed at highest levels in normal prostate. All five Gst genes showed greatly reduced expression in primary tumors compared to normal prostate, but not in tumor metastases. Gst promoter methylation was unchanged in TRAMP tumors compared to normal prostate. Combined decitabine + TSA treatment significantly enhanced the expression of 4/5 Gst genes in TRAMP-C2 cells. CONCLUSIONS: Gst genes are extensively downregulated in primary but not metastatic TRAMP tumors. Promoter DNA hypermethylation does not appear to drive Gst gene repression in TRAMP primary tumors; however, pharmacological studies using TRAMP cells suggest the involvement of epigenetic mechanisms in Gst gene repression.