B-cell non-Hodgkin Lymphoma Cell Lines Research Articles

The Potent and Selective Antitumor Activity of CO-1 and Fully Humanized CO-1bi in Treatment of B Cell Non-Hodgkin's Lymphoma

Recent advancements have identified CD47 blockade as a promising approach for the treatment of B cell Non-Hodgkin's Lymphoma (B-NHL). CD47 blockade inhibits the CD47-SIRPα axis, which inhibits a “don't eat me” signal that facilitates the phagocytosis of tumor cells by macrophages [1,2]. While the effect of anti-CD47 monoclonal antibodies (mAbs) as monotherapy has been limited in B-NHL, combining CD47 mAbs with conventional treatment such as rituximab has demonstrated more encouraging outcomes. This combinatory approach may be attributed to the need for additional cytotoxic mechanisms to achieve clinically meaningful responses with CD47-mediated therapy in B-NHL [3]. Additionally, CD47 blockage has raised concerns about off-target effects and red blood cell depletion [4]. To address these challenges and improve treatment possibilities we developed a fully humanized CO-1bi based on the chimeric CO-1. The bifunctional CO-1 is an agonistic IgG4 anti-CD47 mAb designed to induce direct and rapid programmed cancer cell death (PCCD) and stimulate phagocytosis through blockage of CD47-SIRPα binding. CO-1bi is a humanized bivalent scFv-Fc fusion of CO-1 with superior safety (no red blood cell hemagglutination) while maintaining the functionality of CO-1. In this study, we evaluated the antitumor activity of CO-1 and CO-1bi on different B-NHL cell lines in vitro as well as in xenografts of B-NHL established in NOD- scid IL2Rγ null (NSG) mice. Both constructs demonstrated potent and fast cell death induction in lymphoma cell lines while sparing normal B cells. The robust antitumor activity demonstrated by CO-1 and CO-1bi as single agents in xenograft models of Burkitt's lymphoma underscores the significance of their additional agonistic PCCD capabilities compared to other anti-CD47 agents. The emergence of CO-1bi offers a unique strategy with dual functionality for the treatment of B-NHL and other malignancies without red blood cell depletion characterized by current anti-CD47 mAb in clinical trials. This innovative approach provides an improved and promising avenue for cancer treatment.

Combination of CDK4/6 Inhibitor Palbociclib and PI3K Inhibitor Idelalisib Synergistically Induces an Anti-Tumor Effect in B-Cell Lymphoma and Overcomes Ibrutinib Resistance

Introduction: Aberrant activity of cyclin-dependent kinases 4/6 (CDK4/6) is frequently detected in B-cell non-Hodgkin's lymphoma (B-NHL), indicating the therapeutic implications of CDK4/6 inhibitors for B-cell malignancies. What's more, the introduction of agents targeting B cell receptor signaling, especially phosphatidylinositol 3-kinase (PI3K) isoform-specific inhibitors, is rapidly changing how B cell malignancies are treated. In this study, we aim to investigate whether the combination of CDK4/6 inhibitor palbociclib (PAL) and PI3Kδ inhibitor idelalisib (IDE) has synergistic anti-tumor effects in B cell lymphoma. Besides, we also evaluated the effect of co-administration PAL and IDE in Bruton's tyrosine kinase inhibitor (BTKi) resistant model. Methods: In vitro, cell viability was analyzed using Cell Titer-Glo ®️ Luminescent Cell Viability Assay. Flow cytometry was used to detect apoptosis and cell cycle arrest induction. Western Blotting analysis was used to detect the changes in essential proteins in related signaling pathways. We also conducted RNA-seq to evaluate the whole transcriptome changes brought by co-treatment with PAL and IDE. The synergistic anti-tumor effects of PAL and IDE were also evaluated in vivo. BTKi secondary resistant cell lines were generated by long-time exposure to ibrutinib and BTK C481S mutation cell line was conducted by lentivirus infection and puromycin selection. Results: Firstly, we analyzed diffuse large cell lymphoma (DLBCL) gene expression profile results from GEO database, and there were more than 50% patients carried cell cycle-related genes alterations, especially for CDKN2A-CDK4/6/CCND1-Rb machinery. Then, it was noteworthy that the high level of CDK4/6 mRNA and protein expression correlated with worse survival of DLBCL patients. Next, we illustrated that pharmacological inhibition of CDK4/6 kinase activity induced inhibition of cell proliferation, tumor cell apoptosis, and cell senescence. Meanwhile, PAL treatment upregulated the expression of PI3Kδ, p-AKT (S473), and p-mTOR (S2448), suggesting compensatory activation of the PI3K-AKT-mTOR signaling pathway. Thus, combination treatment of PAL and PI3K inhibitor IDE was assessed, and this combination synergistically induced a more potent anti-proliferative effect in several B-NHL cell lines, including mantle cell lymphoma cell lines (Z-138 and Jeko-1), DLBCL cell lines (U2932 and OCI-Ly8) and Burkitt lymphoma cell line (Raji). Meanwhile, co-administration treatment could also trigger synergistic anti-tumor activity in BTK inhibitor ibrutinib-resistant HBL-1 BTK C481S and HBL-1 IR cells (long time exposure to ibrutinib). Next, the flow cytometry results indicated that the B-NHL cells became apoptotic after the PAL and IDE treatment. The activation of apoptosis-related proteins (cleaved-caspase3 and cleaved-PARP) and decrease of anti-apoptosis proteins expression (BCL-2, BCL-xL, XIAP, and MCL-1) were induced by combination of CDK4/6 and PI3K inhibitor treatment. The combination of PAL and IDE also substantially increased the cell population of G0/G1 phase and elevated expression of CDK4, CDK6, and CyclinD1. Furthermore, the gene expression profile analysis demonstrated that the mRNA expression level of PLK1 was significantly decreased by co-administration treatment, rather than in PAL or IDE treatment alone. Finally, the combination treatment also showed synergistic anti-tumor activity in multiple tumor-bearing mice models. Conclusion: The combination of CDK4/6 inhibitor palbociclib and PI3K inhibitor idelalisib synergistically induced anti-tumor activity in B-cell lymphoma through downregulation of PLK1 expression, suggested a new combination direction for the treatment of B-NHL and even BTK inhibitor-resistant patients.

PROTEOMIC PROFILING IDENTIFIES GRANZYME B INHIBITOR SERPIN B9 AS MEDIATOR OF RESISTANCE TO CAR T‐CELL AND BISPECIFIC ANTIBODY TREATMENT IN NODAL B‐CELL LYMPHOMA

Introduction: Treatment options for relapsed or refractory (r/r) B-cell non-Hodgkin lymphomas (B-NHL) have broadened towards T-cell engaging therapies, including CD19-targeting chimeric antigen receptor T-cells (CD19-CAR) and bispecific antibodies (CD19-BsAb). Although CD19-CAR and CD19-BsAb induce durable responses in some r/r B-NHL patients, response remains heterogenous and a significant proportion of patients experience insufficient responses or relapse. A thorough understanding of tumor-intrinsic mechanisms of resistance is crucial for the clinical improvement of T-cell immunotherapy. Methods: Aiming to identify lymphoma cell-inherent mechanisms that impair response to T-cell engaging therapy, we quantified the in-vitro response of 46 B-NHL cell lines to 3rd generation CD19-CAR and CD19-BsAb. Response was measured using a high-throughput flow cytometry-based assay. In parallel, cell lines were subjected to proteomic profiling and protein abundance was regressed on in-vitro response in order to identify proteomic determinants of response. We further combined CD19-CAR treatment with clinically relevant drugs and assessed how killing of lymphoma cells and expansion of T-cells was altered. Results: Response to CD19-CAR and CD19-BsAb was highly heterogeneous across cell lines and B-NHL entities, but not significantly linked to their proliferation rate or CD19 expression. Integration of the proteomic profiles revealed that abundance of Serpin B9, endogenous inhibitor of T-cell effector molecule granzyme B, was associated with poor response to CD19-CAR and CD19-BsAb. This finding was validated in overexpression and knock-out models. Overexpression of SERPINB9 indeed attenuated response to CD19-CAR and CD19-BsAb, while its knock-out rendered cell lines more sensitive. The observed changes in susceptibility to T-cell-mediated cytotoxicity upon modulation of SERPINB9 expression were independent of T-cell donor and expression of T-cell activation and proliferation markers. We sought to improve T-cell-mediated cytotoxicity specifically in samples with high SERPINB9 expression and found that immune checkpoint inhibitors and lenalidomide enhanced expansion of CAR T-cells and increased killing of lymphoma cells. In addition, vorinostat and antibody-drug-conjugate polatuzumab vedotin specifically killed lymphoma cells without affecting T-cell expansion. However, the observed improvement in T-cell-mediated cytotoxicity was independent of SERPINB9 expression. Keywords: aggressive B-cell non-Hodgkin lymphoma, immunotherapy, indolent non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.

G protein-coupled receptor 183 mediates the sensitization of Burkitt lymphoma tumors to CD47 immune checkpoint blockade by anti-CD20/PI3Kδi dual therapy.

Immunotherapy-based regimens have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decades; however, most disease subtypes remain almost incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory (R/R) B-NHL patients either as a single-agent or in combination with ublituximab, a new generation CD20 antibody. A set of eight B-NHL cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells, M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 alone or combined with the U2 regimen associating ublituximab to the PI3Kδ inhibitor umbralisib, was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and/or quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). CRISPR-Cas9 gene edition was used to selectively abrogate GPR183 gene expression in B-NHL cells. In vivo, drug efficacy was determined in immunodeficient (NSG mice) or immune-competent (chicken embryo chorioallantoic membrane (CAM)) B-NHL xenograft models. Using a panel of B-NHL co-cultures, we show that TG-1801, by disrupting the CD47-SIRPα axis, potentiates anti-CD20-mediated ADCC and ADCP. This led to a remarkable and durable antitumor effect of the triplet therapy composed by TG-1801 and U2 regimen, in vitro, as well as in mice and CAM xenograft models of B-NHL. Transcriptomic analysis also uncovered the upregulation of the G protein-coupled and inflammatory receptor, GPR183, as a crucial event associated with the efficacy of the triplet combination. Genetic depletion and pharmacological inhibition of GPR183 impaired ADCP initiation, cytoskeleton remodeling and cell migration in 2D and 3D spheroid B-NHL co-cultures, and disrupted macrophage-mediated control of tumor growth in B-NHL CAM xenografts. Altogether, our results support a crucial role for GPR183 in the recognition and elimination of malignant B cells upon concomitant targeting of CD20, CD47 and PI3Kδ, and warrant further clinical evaluation of this triplet regimen in B-NHL.

Galunisertib synergistically potentiates the doxorubicin-mediated antitumor effect and kickstarts the immune system against aggressive lymphoma.

In clinical practice, major efforts are underway to identify appropriate drug combinations to boost anticancer activity while suppressing unwanted adverse effects. In this regard, we evaluated the efficacy of combination treatment with the widely used chemotherapeutic drug doxorubicin along with the TGFβRI inhibitor galunisertib (LY2157299) in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The antiproliferative effects of these drugs as single agents or in combination against several B-NHL cell lines and the synergism of the drug combination were evaluated by calculating the combination index. To understand the putative molecular mechanism of drug synergism, the TGF-β and stress signaling pathways were analyzed after combination treatment. An aggressive lymphoma model was used to evaluate the anticancer activity and post-therapeutic immune response of the drug combination in vivo. Galunisertib sensitized various B-NHL cells to doxorubicin and in combination synergistically increased apoptosis. The antitumor activity of the drug combinations involved upregulation of p-P38 MAPK and inhibition of the TGF-β/Smad2/3 and PI3K/AKT signaling pathways. Combined drug treatment significantly reduced tumor growth and enhanced survival, indicating that the synergism between galunisertib and Dox observed in vitro was most likely retained in vivo. Based on the tumor-draining lymph node analysis, combination therapy results in better prognosis, including disappearance of disease-exacerbating regulatory T cells and prevention of CD8+ T-cell exhaustion by downregulating MDSCs. Galunisertib synergistically potentiates the doxorubicin-mediated antitumor effect without aggravating the toxic effects and the ability to kickstart the immune system, supporting the clinical relevance of targeting TGF-βRI in combination with doxorubicin against lymphoma.

Characterization of anti-CD79b/CD3 bispecific antibody, a potential therapy for B cell malignancies.

High rates of relapse and poor prognosis confer an urgent need for novel therapeutic agents for B cell non-Hodgkin lymphomas (B-NHLs). Herein, we describe a human IgG-like anti-CD79b/CD3 bispecific antibody (IBI38D9-L) that selectively depletes antigen-positive malignant B cells as an alternative treatment option for relapsed or refractory NHL patients. The antitumor activity and mechanism of action of IBI38D9-L were investigated in vitro using B-NHL cell lines and human primary effector cells and in vivo using xenograft models reconstituted with human PBMCs (peripheral blood mononuclear cells). Pharmacokinetic (PK) properties and preclinical toxicology were evaluated in cynomolgus monkeys and HSC-NPG mice. IBI38D9-L exerted potent B cell killing as well as T cell activation and proliferation in a tumor cell-dependent manner in vitro and was active against B-NHL cell lines with various CD79b expression levels. Subcutaneous xenograft tumors in NOG mice engrafted with human PBMCs were eradicated by IBI38D9-L treatment. Moreover, IBI38D9-L-treated mice showed a strong infiltration of activated T cells. In HSC-NPG mice, IBI38D9-L resulted in potent B cell depletion in peripheral blood and induced only slight body weight loss and cytokine release syndrome without significant toxicological findings. In cynomolgus monkeys, IBI38D9-L was well tolerated with good pharmacokinetic profiles. Collectively, these preclinical efficacy and safety data provide strong scientific rationales for using anti-CD79b/CD3 bispecific antibody as a promising therapeutic agent for B cell malignancies.

Abstract 6303: Activity and tolerability of combination of venetoclax with the Aurora kinase B Inhibitor AZD2811 in preclinical diffuse large B-cell lymphoma models

Abstract Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin lymphoma (NHL) and the most common form of adult lymphoma. Despite efforts to develop new therapies, the standard of care for DLBCL remains chemotherapy based. The current standard of care is comprised of a regimen of 4 drugs (cyclophosphamide, doxorubicin, vincristine, and prednisone) combined with immunotherapy using a chimeric mAb against the protein CD20 (rituximab), R-CHOP. Unfortunately only 50 to 60% of patients with DLBCL achieve and maintain complete remission after first-line therapy and 40 to 50 % relapse or have refractory disease. In addition, the average age at the time of diagnosis for DLBCL is 64 which makes intensive chemotherapy challenging. Therefore, the development of new treatment options is critical. We explored preclinical activity of AZD2811 nanoparticle, a potent and selective inhibitor of Aurora B kinase combined with venetoclax, an inhibitor of BCL2 currently used as standard of care in treatment of Haematological cancers, in B-Cell non-Hodgkin lymphoma models. Twenty five B-cell NHL cell lines including 11 Diffuse Large B-cell Lymphoma (DLBCL) cell lines were exposed to a 7 x 7 matrix of AZD2811 and venetoclax for 72 hrs. Cell viability was assessed using Celltiter-glo. Strong combination activity (HSA > 0.1 and combination Emax > 0.5) was observed in 6 cell lines including 2 DLBCL cell lines (WSU-DLCL2 and KARPAS_422). Moreover, pre-treatment with pan caspase inhibitor Q-VD-OPH, increased cell viability in cells treated with the combination. Next we explored the significant gene expression and mutation biomarkers (effect size > 1, p < 0.05) for this combination associated with Bliss and combination Emax in B-cell NHL cell lines. We found downregulation of 4 genes (FLT4, TIE1, PRKN) and mutations in 2 genes (EP300 and PTEN) to be associated with Bliss score and upregulation of one gene (FGR) associated with combination Emax. In vivo, antitumor activity of AZD2811 combined with venetoclax was assessed in mice bearing WSU-DLCL2luc xenografts. Once weekly intravenous administration of 25 mg/kg AZD2811 resulted in a statistically significant tumor growth inhibition (TGI) of 74%, while daily oral gavage of 100 mg/kg venetoclax resulted in 49% TGI but failed to reach statistical significance. While both monotherapies were unable to prevent progressive tumor growth, a combination dosing regimen of AZD2811 and venetoclax drove progressive tumor regression resulting in statistically significant complete regression (98% regression) by the third week of dosing. These results suggest combining AZD2811 with venetoclax has potential to be active in DLBCL patients. Citation Format: Azadeh Cheraghchi-Bashi, Brandon Willis, Kevin. Mongeon, Jacob Gordon, Krishna Bulusu, Elizabeth Coker, Patricia Jaaks, Mathew Garnett, Jerome Mettetal. Activity and tolerability of combination of venetoclax with the Aurora kinase B Inhibitor AZD2811 in preclinical diffuse large B-cell lymphoma models [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 6303.

Abstract CT127: Trial in progress: A phase II, open-label, multicenter study of capivasertib, a potent, oral pan-AKT inhibitor in patients with relapsed or refractory B-cell non-hodgkin lymphoma (CAPITAL)

Abstract Background: The PI3K/AKT/mTOR pathway is an established therapeutic target in indolent B-cell non-Hodgkin lymphoma (B-NHL). Several PI3K inhibitors are approved to treat relapsed/refractory (R/R) follicular lymphoma (FL) and, more recently, marginal zone lymphoma (MZL). Nonetheless, potential class-specific and PI3K-isoform-related toxicities may limit their clinical utility. Capivasertib is an oral, potent, selective inhibitor of the 3 AKT isoforms that demonstrated a manageable safety profile (n>1500) and survival benefit in combination with other agents in patients (pts) with solid tumors. Unlike PI3K inhibitors, colitis or pneumonitis have not been a concern with monotherapy. Preclinically, capivasertib was active both in vitro in a large panel of B-NHL cell lines and in vivo in PTEN-null GCB-DLBCL PDX models, where it downregulated the oncogenic factor MYC. In the JVM2 mantle cell lymphoma (MCL) cell line, capivasertib showed superior activity vs PI3Ka/d (AZD8835) and PI3Kb/d (AZD8186) inhibitors. Of note, AKT activation following dysregulation of PTEN has been frequently described in FL (where >85% of tFL or FL/DLBCL exhibited a GCB phenotype). Therefore, capivasertib may represent an effective treatment option for pts with R/R B-NHL. Trial Design: This modular phase II study will enroll pts in 3 cohorts according to B-NHL type. Core inclusion criteria include: age ≥18 years and ECOG PS ≤2. Module-specific criteria include: cohort 1A: R/R FL after ≥2 prior systemic lines (max 5), including an anti-CD20 monoclonal antibody (mAb) and an alkylating agent; cohort 1B: R/R MZL of splenic, nodal, and extranodal subtypes after ≥2 prior systemic lines (max 5) including ≥1 anti-CD20 mAb; cohort 1C: R/R MCL after ≥2 prior systemic lines (max 4), previously exposed to anti-CD20 mAb and a BTK inhibitor. Pts with FL grade 3B, transformed disease, blastoid or TP53 mutation MCL, active CNS disease, diabetes requiring insulin, or being <3 months post-CD19-directed CAR-T therapy will be excluded.Capivasertib will be administered orally at 480 mg BID with an intermittent schedule (4 days on/3 days off; 28-day cycles) until disease progression or unacceptable toxicity. The primary efficacy endpoint in AACR 2022_Capivasertib TiP Abstracteach cohort is objective response rate based on 2014 Lugano criteria per blinded independent central review. Secondary endpoints include duration of response, progression-free survival, overall survival, patient-reported outcomes, safety, and pharmacokinetics. An interim analysis of safety will occur when approximately 30 pts across all cohorts have been treated for 8 weeks. An interim analysis of efficacy will occur after 28 pts (cohorts 1A+1B) or 24 pts (cohort 1C) have been treated for 4 months. A total of 272 pts are planned across 28 sites in 7 countries globally (US, Europe, and Asia). Recruitment began in October 2021. Funding: AstraZeneca Clinical Trial Registration: NCT050080552 Citation Format: Michael Wang, Ho-Jin Shin, Alex F. Herrera, Moshe Levy, Wong Seog Kim, Tae Min Kim, Jin Seok Kim, Dok Hyun Yoon, Vincent Ribrag, Miguel Canales, John Radford, Dima El-Sharkawi, Brandy Hemmer, Richa Manwani, Veerendra Munugalavadla, Robert Chen, Andrew Mortlock, Alessandra Forcina, Franck Morschhauser. Trial in progress: A phase II, open-label, multicenter study of capivasertib, a potent, oral pan-AKT inhibitor in patients with relapsed or refractory B-cell non-hodgkin lymphoma (CAPITAL) [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 CT127.

Sting Is Commonly and Differentially Expressed in T- and Nk-Cell but Not B-Cell Non-Hodgkin Lymphomas.

Simple SummaryT/NK-cell non-Hodgkin lymphomas (NHLs) represent approximately 10% of all NHLs and most patients have a poor outcome using current treatment options. Molecules involved in the host response against lymphoma cells are currently being investigated in an effort to develop novel therapeutic strategies combining targeted therapy and immunotherapy. In this study, we show that expression of STING, a key protein in the cGAS–STING immune response pathway, is restricted to lymphomas of T- and NK-cell origin and seems to be down regulated in B-cell NHLs. These results are based on the analysis of 14 lymphoma cell lines of various types at the RNA and protein level and immunohistochemical analysis of a large number of B-cell (n = 265) and T/NK-cell (n = 158) NHLs obtained from previously untreated patients from three institutions. In these patient cohorts, STING is differentially expressed among T/NK-cell NHLs, whereas all B-cell NHLs were negative for STING expression. Thus, STING represents a novel biomarker and therapeutic target in T- and NK-cell lymphomas with direct immunotherapeutic implications, since modulators of cGAS–STING activity are already available for clinical use, and could therefore be used to benefit patients with these difficult-to-treat diseases. The expression patterns of stimulator of interferon genes (STING) were investigated in a cohort of 158 T- and natural killer (NK)-cell and 265 B-cell non-Hodgkin lymphomas (NHLs), as well as in control reactive lymph nodes and tonsils. STING expression was assessed by immunohistochemical methods using diagnostic biopsy specimens obtained prior to treatment. Using an arbitrary 10% cutoff, STING was differentially expressed among T/NK-cell NHLs; positive in 36 out of 38 (95%) cases of ALK+ anaplastic large cell lymphoma (ALCL), 23 out of 37 (62%) ALK-ALCLs, 1 out of 13 (7.7%) angioimmunoblastic T-cell lymphomas, 15 out of 19 (79%) peripheral T-cell lymphomas, not otherwise specified, 20 out of 36 (56%) extranodal NK/T-cell lymphomas of nasal type, 6 out of 7 (86%) T-cell lymphoblastic lymphomas, and 3 out of 4 (75%) mycosis fungoides. STING expression did not correlate with clinicopathological parameters or outcome in these patients with T/NK-cell lymphoma. By contrast, all 265 B-cell NHLs of various types were STING-negative. In addition, STING mRNA levels were very high in 6 out of 7 T-cell NHL cell lines, namely, ALK+ and ALK-ALCL cell lines, and very low or undetectable in 7 B-cell NHL cell lines, suggesting transcriptional downregulation of STING in neoplastic B-cells. At the protein level, using Western blot analysis and immunohistochemistry performed on cell blocks, STING expression was found to be restricted to T-cell NHL cell lines. Taken together, STING expression represents a novel biomarker and therapeutic target in T- and NK-cell lymphomas with direct immunotherapeutic implications since modulators of cGAS–STING activity are already available for clinical use.

Antitumor Activity of the Novel BTK Inhibitor TG-1701 Is Associated with Disruption of Ikaros Signaling in Patients with B-cell Non-Hodgkin Lymphoma.

Despite the remarkable activity of BTK inhibitors (BTKi) in relapsed B-cell non-Hodgkin lymphoma (B-NHL), no clinically-relevant biomarker has been associated to these agents so far. The relevance of phosphoproteomic profiling for the early identification of BTKi responders remains underexplored. A set of six clinical samples from an ongoing phase I trial dosing patients with chronic lymphocytic leukemia (CLL) with TG-1701, a novel irreversible and highly specific BTKi, were characterized by phosphoproteomic and RNA sequencing (RNA-seq) analysis. The activity of TG-1701 was evaluated in a panel of 11 B-NHL cell lines and mouse xenografts, including two NF-κB- and BTKC481S-driven BTKi-resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, Western blot analysis, immunostaining, and gene knockout (KO) experiments. A nonsupervised, phosphoproteomic-based clustering did match the early clinical outcomes of patients with CLL and separated a group of "early-responders" from a group of "late-responders." This clustering was based on a selected list of 96 phosphosites with Ikaros-pSer442/445 as a potential biomarker for TG-1701 efficacy. TG-1701 treatment was further shown to blunt Ikaros gene signature, including YES1 and MYC, in early-responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and signaling remained unaffected by the drug in vitro and in vivo in late-responder patients and in BTKC481S, BTKKO, and noncanonical NF-κB models. These data validate phosphoproteomic as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of drug mechanism of action.

High Frequency of CD74 Expression in B-Cell Non-Hodgkin's Lymphoma (NHL) and Targeting with SΤRO-001, a Novel Anti-CD74 Antibody Drug Conjugate (ADC) with Potent I n Vitro Cytotoxicty and In Vivo Anti-Tumor Activity

Introduction: STRO-001 is a novel CD74-targeting ADC comprised of an anti-CD74 human IgG1 antibody (SP7219) genetically incorporating the non-natural amino acid para-azidomethyl-L-phenylalanine (pAMF) with a non-cleavable dibenzocyclooctyne (DBCO)-maytansinoid linker-warhead. Highly efficient and precise site-specific conjugation enabled by cell-free antibody synthesis technology produced a well-defined homogeneous ADC with a drug-antibody ratio (DAR) of 2. Conjugation sites were selected based on highest stability both in vitro and in vivo . The aim of this study was to investigate the expression of CD74 in NHL and explore the therapeutic potential of STRO-001 in B-cell NHL cell lines and xenografts.Methods: The binding affinity of SP7219 to recombinant human CD74 ECD (extracellular domain) and cynomolgus monkey (cyno) CD74 ECD was measured by SPR using a Biacore T200. Biotinylated SP7219 was used for immunohistochemistry (IHC) in NHL tumor specimens. Flow cytometry was used for measuring CD74 expression in NHL cell lines. STRO-001 was used to determine the EC50 and percent span of killing in NHL cell lines. The anti-tumor activity of STRO-001 in NHL xenografts in severe combined immune deficient (SCID) mice was examined.Results: Anti-CD74 antibody SP7219 binds to both human and cyno CD74 ECD with high affinity (KD=0.85nM for human, KD=2.4nM for cyno). Tissue samples from the Department of Pathology at Stanford University were used to evaluate CD74 expression by IHC after appropriate permissions were obtained. Medium to high CD74 expression in >70% of cells was observed in 118/118 (100%) diffuse large B-cell lymphoma (DLBCL), 148/151 (98%) follicular lymphoma and 19/21 (90%) mantle cell lymphoma (MCL) samples. In vitro cytotoxicity assays show potent activity of STRO-001 in a diverse panel of B-cell tumor lines with EC50 values ranging from 0.17-13 nM (Table 1). CD74 cell surface expression is required for STRO-001 cytotoxicity but expression level does not correlate highly with in vitro potency (R2 = 0.4154). STRO-001 exhibits dose-dependent tumor growth inhibition in rituximab-resistant germinal center B-cell-like (GCB)-DLBCL SU-DHL-6 xenografts starting at 2.5 mg/kg weekly x 3 doses. STRO-001 + bendamustine/rituximab (BR) further improves tumor suppression in SU-DHL-6 xenografts compared to vehicle (p = 0.002) or BR alone (p = 0.02). In the activated B-cell (ABC)-DLBCL U2932 model, single doses of STRO-001 produce dose-dependent growth inhibition at 1 and 3 mg/kg and complete tumor regression at 10 mg/kg in 7/7 animals with no tumor re-growth up to > 90 days post-treatment (Figure 1). Studies with an MCL model, Jeko-1, demonstrate robust STRO-001 anti-tumor activity compared to vehicle (p < 0.0001) starting at a single 3 mg/kg dose, with a single 10 mg/kg dose resulting in tumor regression for up to 64 days post treatment (Figure 2). STRO-001 treatment 14 days post tumor inoculation was used to evaluate disease progression in the disseminated Mino MCL model. Vehicle-treated animals developed advanced progressive disease, with median survival of 81 days. In contrast, all Mino xenografts treated with STRO-001 at 3 mg/kg or 10 mg/kg were alive and disease-free at the time of sacrifice 135 days post tumor inoculation (Figure 3).Conclusions: CD74 is expressed with high frequency and intensity in DLBCL, follicular lymphoma and MCL, supporting the investigation of novel biologics targeting this antigen. STRO-001 demonstrates potent in vitro cytotoxicity in multiple NHL cell lines and anti-tumor activity in GCB-DLBCL, ABC-DLBCL and mantle cell lymphoma xenograft models, including prolongation of survival in the disseminated Mino MCL model and prevention of tumor regrowth in the U2932 ABC-DLBCL and Jeko-1 MCL subcutaneous models. Clinical studies of this novel ADC for treatment of B-cell malignancies are under development. [Display omitted] DisclosuresYu:Sutro Biopharma: Employment. Abrahams:Sutro Biopharma: Employment. Embry:Sutro Biopharma: Employment. Li:Sutro Biopharma: Employment. Zhao:Stanford University School of Medicine: Employment. Henningsen:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Natkunam:Stanford University School of Medicine: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment.

PD-1 blockade enhances cytokine-induced killer cell-mediated cytotoxicity in B-cell non-Hodgkin lymphoma cell lines.

The clinical utility of immune checkpoint inhibitors, such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors used alone or in combination with other therapies, is currently gaining attention. In this particular scenario, the inclusion of cytokine-induced killer (CIK) cells has proven to be a novel therapeutic approach. CIK cells have shown anticancer activity in various hematopoietic malignancies, but their defined cytotoxicity in B-cell non-Hodgkin lymphoma (B-NHL) remains to be fully elucidated. The present study investigated the role of PD-1/PD-L1 blockades on the cytotoxic efficacy of CIK cells primarily in B-NHL cell lines. The current analysis revealed that CIK cells prompted cytotoxicity against B-NHL cell lines (DAUDI and SU-DHL-4), and a significant increase in PD-L1 expression was observed when CIK cells were co-cultured with B-NHL cells. Additionally, a combination of PD-1 and PD-L1 antibodies with CIK cells significantly decreased cell viability only in DAUDI cells. Furthermore, IFN-γ elevation was observed in both cell lines treated with CIK alone or with PD-1 antibody, but this tendency was not observed for PD-L1. Since PD-1 can suppress immune inactivation, whereas CD40L can promote it, the effects of CD40L blockade were also examined; however, no significant changes in cell viability were observed. Overall, the present in vitro data suggested that CIK cells exerted a cytotoxic function in B-NHL cells, and a combination of PD-1 inhibitors with CIK cells may provide a potential therapeutic option for this type of lymphoma. Nevertheless, in vivo experiments are warranted to undermine the extent to which PD-1 inhibitors may be used to enhance the antitumor activity of CIK cells in B-NHL.

Preclinical Anti-Tumor Activity of Hexabody-CD38 in Patient-Derived B Cell Lymphoma and Acute Myeloid Leukemia Xenograft Models

HexaBody®-CD38 (GEN3014) is a next-generation CD38-specific IgG1 molecule with a hexamerization-enhancing mutation that leads to highly efficient induction of complement-dependent cytotoxicity (CDC) upon binding to CD38 positive tumor cells. HexaBody-CD38 is designed to induce strong anti-tumor activity in patients with CD38-expressing hematological malignancies through CDC and other Fc-mediated effector functions. Previously we have shown that HexaBody-CD38 induces highly potent CDC-mediated cell death in multiple myeloma (MM), acute myeloid leukemia (AML), and B-cell non-hodgkin lymphoma (B-NHL) cells, including tumor cells in bone marrow samples from MM patients. In addition, HexaBody-CD38 demonstrated efficient ADCC and ADCP and induction of direct cell kill after Fc-crosslinking. Moreover HexaBody-CD38 induced strong inhibition of CD38 cyclase activity, which may contribute to reduction of immune suppression in the tumor microenvironment. In this study, we show that in a panel of 23 MM, AML and B-NHL cell lines HexaBody-CD38 induced CDC, with EC50 values on average 7-fold lower than those of the CD38-targeting mAb daratumumab, which is part of the standard of care for MM. Cytotoxic activity of HexaBody-CD38 showed a positive correlation with CD38 expression levels. HexaBody-CD38 was especially more potent than daratumumab in cell lines with lower CD38 expression. Sensitivity to HexaBody-CD38-induced CDC was correlated with lower expression levels of complement regulatory proteins, including CD46, CD55 and CD59. To exclude that the enhanced potency of HexaBody-CD38 resulted in lysis of healthy cell subsets known to express low levels of CD38, CDC in healthy donor leukocytes was assessed in vitro. HexaBody-CD38 (20 μg/mL) did not induce lysis of monocytes, B cells, T cells, granulocytes or erythrocytes, and only minor cytotoxicity of NK cells (median 13% lysis, range 9.5-55.1%). NK-cell lysis by daratumumab, assessed in parallel, was in the same range (median 8%, range 4.5-21.2%). Thus, HexaBody-CD38 shows superior potency in CD38-positive tumor cells while sparing healthy donor leukocytes and erythrocytes in vitro. The anti-tumor activity of HexaBody-CD38 in vivo was evaluated in patient-derived AML and B-NHL xenograft (PDX) models in nude mice. In a screen using 9 B-NHL PDX models in a 1-3 mice-per-treatment-group design, administration of two weekly doses of 5 mg/kg HexaBody-CD38 induced a strong anti-tumor response (relative tumor growth ≤10%, i.e. tumor stasis or tumor regression) in 2 models. Two models were classified as non-responder (relative tumor growth &amp;gt;70%) and 5 models could not be classified as either responder or non-responder (intermediates, relative tumor growth between 10 and 70%). A dose-response experiment in one of the responding models (Ly12638, 9 mice per group), confirmed potent anti-tumor activity of HexaBody-CD38, inducing complete tumor regression at a dose of 10 mg/kg (Figure 1A). In a screen using 5 AML PDX models in a 1-3 mouse-per-treatment-group design, HexaBody-CD38 (5 mg/kg) induced a strong anti-tumor response in one model. One model was classified as non-responder and the remaining 3 models could not be classified as either responder or non-responder (intermediates). A dose-response experiment in one of the intermediate models (AML11810), demonstrated statistically significant and dose-dependent anti-tumor activity of HexaBody-CD38 in an in vivo study using a classical 9 mice per treatment group design (Figure 1B). In summary, HexaBody-CD38 induced potent CDC in MM, B-NHL and AML cell lines, with superior potency compared to benchmark antibody daratumumab. HexaBody-CD38 was able to induce CDC in tumor cell lines with lower levels of CD38 expression, while sparing healthy leukocyte subsets. HexaBody-CD38 showed potent anti-tumor activity in vivo both in B-NHL and AML PDX models in nude mice. In these models, the contribution of mouse complement to the anti-tumor activity is limited and therapeutic activity is thought to be driven mainly through FcγR-mediated effector functions. This indicates that besides highly potent CDC, FcɣR-mediated effector mechanisms contribute to the preclinical activity of HexaBody-CD38 in hematological tumor models. These preclinical data support clinical investigation of HexaBody-CD38 in CD38 positive hematologic malignancies, including MM, AML, and B cell lymphomas. Figure Disclosures Hiemstra: Genmab: Current Employment, Current equity holder in publicly-traded company. Janmaat:Genmab: Current Employment, Current equity holder in publicly-traded company. Boross:Genmab: Current Employment, Current equity holder in publicly-traded company. van den Brakel:Genmab: Current Employment, Current equity holder in publicly-traded company. ten Hagen:Genmab: Current Employment, Current equity holder in publicly-traded company. van Dooremalen:Genmab: Current Employment, Current equity holder in publicly-traded company. Bosgra:Genmab: Current Employment, Current equity holder in publicly-traded company. De Goeij:Genmab: Current Employment, Current equity holder in publicly-traded company. Andringa:Genmab: Current Employment, Current equity holder in publicly-traded company. Kil:Genmab: Current Employment, Current equity holder in publicly-traded company. Sasser:Genmab: Current Employment, Current equity holder in publicly-traded company. Ahmadi:Genmab: Current Employment, Current equity holder in publicly-traded company. Satijn:Genmab: Current Employment, Current equity holder in publicly-traded company. Breij:Genmab: Current Employment, Current equity holder in publicly-traded company.

The B-cell receptor in control of tumor B-cell fitness: Biology and clinical relevance.

Surface expression of a functional B cell antigen receptor (BCR) is essential for the survival and proliferation of mature B cells. Most types of B-cell lymphoproliferative disorders retain surface BCR expression, including B-cell non-Hodgkin lymphomas (B-NHL) and chronic lymphocytic leukemia (CLL). Targeting BCR effectors in B-NHL cell lines in vitro has indicated that this signaling axis is crucial for malignant B cell growth. This has led to the development of inhibitors of BCR signaling, which are currently used for the treatment of CLL and several B-NHL subtypes. Recent studies based on conditional BCR inactivation in a MYC-driven mouse B-cell lymphoma model have revisited the role of the BCR in MYC-expressing tumor B cells. Indeed, lymphoma cells losing BCR expression continue to grow unless subjected to competition with their BCR-expressing counterparts, which causes their elimination. Here, we discuss the molecular nature of the fitness signal delivered by the BCR to MYC-expressing malignant B cells, ensuring their preferential persistence within a rapidly expanding tumor population. We also review growing evidence of Ig-negative cases belonging to several B-NHL subtypes and CLL, and discuss the clinical implications of these findings in relation to an emerging picture of clinical resistances to anti-BCR therapies.

Suitability of Yin Yang 1 transcript and protein levels for biomarker studies in B cell non-Hodgkin lymphoma

BackgroundYin Yang 1 (YY1) is a transcription factor that plays an important role during all stages of B cell differentiation. Several studies reported upregulation of YY1 in B cell derived lymphoma, indicating that it might act as an oncogene. Furthermore, aberrant YY1 expression has been associated with survival in some entities of B cell non-Hodgkin lymphoma (B-NHL), suggesting that YY1 could be a valuable biomarker in B-NHL. However, studies are controversial and methodologically disparate, partially because some studies are based on transcript levels while others rely on YY1 protein data. Therefore, we aimed to investigate the dependence of YY1 protein levels on YY1 transcription.MethodsA panel of human cell lines representing different B-NHL subtypes was used to test for the correlation of YY1 mRNA and protein levels which were determined by quantitative PCR and immunoblotting. To analyze YY1 mRNA and YY1 protein stability cells were treated with actinomycin-D and cycloheximide, respectively. siRNAs were transfected to knockdown YY1. Kaplan-Meier survival analyses were performed with data from published patient cohorts. Pearson’s correlation analyses were assessed and statistical power was examined by Student’s t-test.ResultsIn the analyzed panel of B-NHL cell lines YY1 transcript levels do not correlate with their cellular protein amounts. YY1 protein levels were unaffected by transient block of transcription or by targeting YY1 mRNA using siRNA. Additionally, global inhibition of translation up to 48 h did not alter protein levels of YY1, indicating that YY1 is a highly stable protein in B-NHL. Furthermore, in a retrospective analysis of two different B-NHL cohorts, YY1 transcript levels had no impact on patients’ survival probabilities.ConclusionsOur results point out the necessity to focus on YY1 protein expression to understand the potential role of YY1 as an oncogene and to unravel its suitability as clinical biomarker in B-NHL.

Pan-class I\xa0 PI3-kinase inhibitor BKM120 induces MEK1/2-dependent mitotic catastrophe in non-Hodgkin lymphoma leading to apoptosis or polyploidy determined by Bax/Bak and p53

Constitutive signaling of PI3K/Akt/mTOR plays a prominent role in malignant transformation and progression of B-cell non-Hodgkin lymphomas (B-NHL) underscoring the need for PI3K targeted therapies. The pan-class I PI3-kinase inhibitor BKM120 has shown preclinical activity in distinct malignancies and is currently tested in clinical trials. Intratumor heterogeneity is an intrinsic property of cancers that contributes to drug resistance and tumor recurrence. Here, we demonstrate that inhibition of PI3-kinases by BKM120 attenuates growth and survival of B-NHL cell lines by inducing mitotic arrest with subsequent induction of intrinsic apoptosis. BKM120-mediated downregulation of Cyclin A and activation of the CDK1/Cyclin B1 complex facilitates mitotic entry. In addition, concomitant BKM120-mediated upregulation of Cyclin B1 expression attenuates completion of mitosis, which results in mitotic catastrophe and apoptotic cell death. In Bax and Bak deficient B-NHL, which are resistant to BKM120-induced apoptosis, BKM120-induced mitotic catastrophe results in polyploidy. Upon re-expression of wt p53 in these p53 mutated cells, BKM120-induced polyploidy is strongly reduced demonstrating that the genetic status of the cells determines the outcome of a BKM120-mediated pathway inhibition. Mitotic catastrophe and unfavorable induction of polyploidy can be prevented in this setting by additional inhibition of MEK1/2 signaling. Combining MEK1/2 inhibitors with BKM120 enhances the anti-tumor effects of BKM120, prevents prognostic unfavorable polyploidy and might be a potential strategy for the treatment of B-NHL.

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas.

Constitutive B-cell receptor signaling leads to overexpression of the antiapoptotic BCL-2 protein and is implicated in the pathogenesis of many types of B-cell non-Hodgkin lymphoma (B-NHL). The BCL-2 small-molecule inhibitor venetoclax shows promising clinical response rates in several lymphomas, but is not curative as monotherapy. Radiotherapy is a rational candidate for combining with BCL-2 inhibition, as DNA damage caused by radiotherapy increases the activity of pro-apoptotic BCL-2 pathway proteins, and lymphomas are exquisitely sensitive to radiation. We tested B-NHL responses to venetoclax combined with either external beam radiotherapy or radioimmunotherapy (RIT), which joins the selectivity of antibody targeting with the effectiveness of irradiation. We first tested cytotoxicity of cesium-137 irradiation plus venetoclax in 14 B-NHL cell lines representing five lymphoma subtypes. Combination treatment synergistically increased cell death in 10 of 14 lines. Lack of synergy was predicted by resistance to single-agent venetoclax and high BCL-XL expression. We then assessed the efficacy of external beam radiotherapy plus venetoclax in murine xenograft models of mantle cell (MCL), germinal-center diffuse large B-cell (GCB-DLBCL), and activated B-cell (ABC-DLBCL) lymphomas. In each model, external beam radiotherapy plus venetoclax synergistically increased mouse survival time, curing up to 10%. We finally combined venetoclax treatment of MCL and ABC-DLBCL xenografts with a pretargeted RIT (PRIT) system directed against the CD20 antigen. Optimal dosing of PRIT plus venetoclax cured 100% of mice with no detectable toxicity. Venetoclax combined with radiotherapy may be a promising treatment for a wide range of lymphomas Cancer Res; 77(14); 3885-93. ©2017 AACR.

CD19 Target Activated Natural Killer (CD19.TaNK) Cellular Therapy: A Novel immunotherapeutic Approach to the Treatment of Non-Hodgkin Lymphoma (NHL)

Background: Continued improvement in the treatment of NHL is desired, especially via the incorporation of 'targeted' immunotherapy agents. This is especially important in B-cell NHL (bNHL) as resistance to rituximab anti-CD20 antibody, and now second-generation antibodies (e.g., obinutuzumab), may occur. Activated NK-92 (aNK-92) is a continuously growing cell line consisting of "pure" (100%) activated NK cells. These cells were subsequently bioengineered to express human anti-CD19 chimeric antigen receptor (CAR) recognizing CD19+ B cells. The goal of this project was to investigate the specificity and the efficacy of a novel 'off the shelf' targeted immunotherapy, CD19.TaNK, in a multitude of B-cell NHL cell lines, including anti-CD20 antibody resistant cell lines.Methods: Using gene expression profiling, Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, we first investigated the expression of NK activation and inhibitory ligands in varied lymphoma cells. The bNHL cell lines, SUDHL10 (DLBCL), L540, L428 (Hodgkin lymphoma), HF1 (follicular), Raji (Burkitt's), and Mino (mantle cell) were purchased from ATCC and maintained in RPMI1640 medium. aNK and CD19.TaNK were supplied by NantKwest, Inc and were maintained in Myelocult supplemented with recombinant human IL-2 (500IU/ml). NK cell mediated cytotoxicity was determined using lactate dehydrogenase (LDH) release glucose-6-phosphate dehydrogenase (G6PD) release (aCella-tox assay). Briefly, 10,000 target bNHL cells were co-cultured with effector NK cells, at clinically relevant effector to target ratios (E:T 1:1-10:1) for 4 hours, and the supernatant was assayed for LDH or G6PD release. Percent cytotoxicity was determined based on the experimental levels of LDH or G6PD release from NK mediated bNHL cell lysis compared to maximum LDH or G6PD release from target cells. To determine if resistance to anti-CD20 antibodies would interfere with sensitivity to CD19.TaNK therapy, rituximab and obinutuzumab resistant bNHL cell lines (SUDHL4, SHUDHL10, and Raji) were established; cells were exposed to incremental increasing concentrations of antibody drugs (5-20μg/ml) over a period of 8 weeks. CD19, CD20 and CD30 expression in bNHL cells was determined by flow cytometry. Additionally, the efficacy of primary NK cells were determined against CD20 monoclonal antibody sensitive and resistant cell lines utilizing droplet microfluidics based assessment.Results: We observed that bNHL cell lines expressed a multitude of ligands associated with stimulating NK cell activity, while expression of inhibitory ligands was minimal. This indicates that NK cell interaction with bNHL cells is predicted to lead to overall robust antitumor immune response (Figure). Using LDH and G6PD release assays in bNHL cell lines, we observed increased cytolytic activity in an E:T ratio dependent manner, with Raji and L428 cells being the most sensitive to CD19.TaNK at 1:1 E:T ratio. Development of resistance to anti-CD20 antibodies (rituximab and obinutuzumab) resulted in significantly decreased down regulation of CD20, but not CD19 or CD30, as detected by flow cytometry. After direct contact with primary NK cells, we observed that rituximab resistant SUDHL10 cells were poorly sensitive (7%), while in rituximab sensitive cells, there was 22% cell loss. Moreover, at 4 hours using CD19.TaNK therapy (1:5 ratios), there was marked cytolytic activity with consistent high LDH release seen across all bNHL cell lines without differences noted regardless of rituximab or obinutuzumab resistance (ie, SUDHL4, SHUDHL10, and Raji). These results were further confirmed using live cell video microscopy measuring the cytolytic activity of CD19.TaNK versus bNHL cells.Conclusion: We identified that bNHL cells contain high expression levels of NK activation ligands and low amounts of inhibitory ligands and that CD19.TaNK immunotherapy had potent single-agent anti-tumor activity against a spectrum of bNHL cells. Furthermore, CD19.TaNK maintained high cytolytic activity in bNHL cells resistant to standard CD20 antibody therapy, which were poorly sensitive to innate NK cells. Ongoing analyses include systems biology studies to determine potential biologic mechanisms of activity of CD19.TaNK therapy as well as well as to help guide optimum combinatorial therapy. [Display omitted] DisclosuresBoissel:NantKwest, Inc.: Employment. Evens:Takeda: Other: Advisory board.

PF-04691502, a dual PI3K/mTOR inhibitor has potent pre-clinical activity by inducing apoptosis and G1 cell cycle arrest in aggressive B-cell non-Hodgkin lymphomas.

The PI3K/Akt/mTOR pathway is activated in a variety of human tumors including B-cell non-Hodgkin lymphoma (B-NHL). Targeting this pathway has been validated in solid and hematological tumors. In the present study, we demonstrated that PF-04691502, a novel PI3K/mTOR inhibitor has potent activity in a panel of aggressive B-NHL cell lines including diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). MTS analysis showed that PF-04691502 effectively inhibited cell proliferation with IC50 values ranging from 0.12 to 0.55µM. Cells treated with PF-04691502 exhibited decreased phosphorylation of Akt and S6 ribosomal protein confirming the mechanism of action of a PI3K/mTOR inhibitor. Also, treatment of B-NHL cell lines with PF-04691502 induced apoptosis in a dose- and time-dependent manner. Moreover, PF-04691502 significantly induced G1 cell cycle arrest associated with a decrease in cyclinD1 which contributed to suppression of cell proliferation. Finally, rituximab enhanced apoptosis induced by PF-04691502. Taken together, our findings provide for the first time that PF-04691502 inhibits the constitutively activated PI3K/mTOR pathway in aggressive B-cell NHL cell lines associated with inhibition of cell cycle progression, cell proliferation and promotion of apoptosis. These findings suggest that PF-04691502 is a novel therapeutic strategy in aggressive B-cell NHL and warrants early phase clinical trial evaluation with and without rituximab.

Chemical Genomics Reveals JAK STAT Activation As a Mechanism of Resistance to HDAC Inhibitors in B Cell Lymphomas

▪Background: HDAC inhibitors (HDACi) are being investigated as treatment for relapsed/refractory non Hodgkin lymphoma (NHL) and other cancers. However, the mechanisms underlying sensitivity and resistance to HDAC inhibition in lymphomas have not been fully characterized. We probed the cellular and molecular response to HDACi in vitro and in vivo in order to determine factors that dictate the response to HDACi and to enable design of approaches to incorporate HDACi into novel combination therapeutics.Methods: High-throughput cytotoxicity screening was performed using two different HDAC inhibitors, LBH589 (panobinostat) and SAHA (vorinostat) in 52 lymphoid cell lines characterized through RNA-seq and microarray gene expression profiling. This screen revealed a greater than 50-fold range in concentration needed to induce cytotoxicity for the 2 different HDAC inhibitors and there was moderate correlation between the 2 compounds in this panel (Pearson correlation r = 0.76, p < 0.01). By pairing this chemosensitivity data with gene expression profiles of the screened cell lines, we developed a gene expression classifier for LBH589 that identified resistant and sensitive cell line groups. This predictor was applied to B-cell NHL cell lines tested with LBH589 in the Cancer Cell Line Encyclopedia (CCLE) and we found that the sensitive and resistant cell line groups distinguished by this method differed more than 5-fold in IC50 (0.021 vs. 1.24 nM, P < 0.01 by Wilcoxon rank sum), thus validating the ability of this approach to distinguish HDACi resistant cell lines.We further initiated a clinical trial of LBH589 in relapsed/refractory diffuse large B cell lymphoma patients combined with RNAseq profiling of their tumors prior to embarking on treatment. We treated nine patients with LBH589, and application of our response predictor to scaled RNAseq gene expression data revealed 4 predicted responders and 5 predicted non-responders. Two of the predicted responders had a clinical response to LBH589, whereas none of the predicted non-responders had a clinical response, thus our classifier was able to identify all of the LBH589-responsive patients from this cohort (P = 0.08 by Fisher’s exact test).Analysis of differentially expressed molecular pathways in HDACi sensitive and resistant samples by gene set enrichment revealed the JAK-STAT pathway as the most differentially expressed pathway associated with HDACi resistance (at P < 0.001 and FDR < 0.20). We further identified a number of distinct mutations including STAT3, SOCS1 and JAK1 that were associated with activation of the JAK-STAT pathway by gene expression signatures and the LBH589 response signature in DLBCL cell lines and patient samples by analysis of RNA-seq data. Phosphoprotein analysis by Western blot and Sis-inducible-element (SIE) luciferase reporter assays were used to confirm JAK-STAT activation in these samples and we found that overexpression of STAT3 Src-homology domain mutations activated JAK-STAT3 signaling in isogenic cell lines and fostered resistance to LBH589 in vitro. Conversely, using in vivo DLBCL xenograft models, we found that combining JAK-STAT and HDAC inhibition by treatment with LBH589 and ruxolitinib resulted in synergistic reduction of tumor cell viability and tumor growth with tolerable toxicity in mice.Conclusions: Sustained JAK-STAT activation appears to mediate resistance to HDAC inhibition in DLBCL and other NHLs and several recurrent genetic lesions drive JAK-STAT activation in these diseases. This process can be overcome by JAK 1/2 inhibition with ruxolitinib and these findings demonstrate a role for combination therapy with HDAC inhibitors and small molecules targeting the JAK-STAT pathway in lymphoid malignancies. DisclosuresNo relevant conflicts of interest to declare.