Reversal of Drug/TRAIL-Resistant B-NHL Cells to Apoptosis by the Combination of Rituximab (anti-CD20) and Either Mapatumumab or Lexatumumab

Abstract Abstract 4931 Conventional treatments of non-Hodgkin's lymphoma (B-NHL) consist primarily of chemotherapy. Currently, rituximab is used alone or in combination with chemotherapy. However, there are subsets of patients who do not respond initially or develop resistance to further treatment. Therefore, there is an urgent need to develop other immunotherapies with less toxicities. At present, both TRAIL and agonist antibodies directed against TRAIL-R1 and -R2 have been explored for various cancer treatments in various phase 1 and phase 2 clinical trials. We have recently demonstrated that rituximab sensitizes TRAIL-resistant B-NHL cells to TRAIL-induced apoptosis. Sensitization was the result of rituximab-induced inhibition of the constitutively activated NF-κB pathway and downstream the DR5 transcription repressor Yin Yang 1 (YY1). The direct role of YY1 in the regulation of resistance to TRAIL was demonstrated in cells transfected with YY1 siRNA and that became sensitive to TRAIL- apoptosis. Treatment with rituximab did not have any observed effects on the expression of DR4. Based on these findings, it was possible that rituximab-mediated sensitization to TRAIL may invoke either TRAIL-R1 (DR4) or TRAIL-R2 (DR5), or both; thus, this possibility is currently being examined by the use of either neutralizing antibodies against each death receptor or by the use of silencing RNA. Currently, clinical trials are being conducted with both mapatumumab (anti-TRAIL-R1,) and lexatumumab (anti-TRAIL-R2) against a variety of cancers. These agonist antibodies have been evaluated clinically as single agents and in combination with standard therapy in solid and hematologic malignancies. It is not clear whether tumors can develop resistance to agonism of either one or both death receptors and thus, may not respond to monotherapy alone. Combination therapies may be required and we have hypothesized that the combination treatment of rituximab and agonist antibodies may be complementary or synergistic. This hypothesis was based on our findings that rituximab inhibits survival pathways and downregulates anti-apoptotic gene products and, thus, significantly reducing the threshold of resistance. Thus, this rituximab-mediated effect will facilitate the direct cytotoxicity of the agonist death receptor antibodies. The present study investigated whether rituximab can sensitize TRAIL-resistant tumor cells by either agonist TRAIL-R1 or TRAIL-R2 antibodies To address this question, we have examined the effect of agonist antibodies directed against either TRAIL-R1 (mapatumumab) or TRAIL-R2 (lexatumamab). Treatment of the TRAIL-resistant Ramos B-NHL cells with rituximab for 24h and followed with treatment with non-toxic concentrations of mapatumumab (12 μg/ml) or lexatumumab (12 μg/ml) for 18h resulted in significant sensitization to apoptosis as assessed by activation of caspase 3. The mechanism of the sensitization by rituximab for each antibody was also examined. These findings demonstrated that rituximab sensitizes tumor cells to apoptosis by activation of either DR4 or DR5. Although there is heterogeneous expression of TRAIL-R1 and TRAIL-R2 in B-NHL cells, such cells may still be sensitive to rituximab-mediated sensitization to apoptosis by the corresponding agonist death receptor antibody. Recent findings demonstrated that some tumors expressing both DR4 and DR5 were shown to respond to TRAIL by preferential activation of DR4 and not DR5. Therefore, preclinical findings obtained with the use of TRAIL may not be predictive of outcome compared to the use of TRAIL-receptor specific agonist antibodies; mapatumumab or lexatumumab. Disclosures: No relevant conflicts of interest to declare.

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Self-Replicating RNA Viruses for RNA Therapeutics

Molecules ◽

10.3390/molecules23123310 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3310 ◽

Cited By ~ 13

Author(s):

Kenneth Lundstrom

Keyword(s):

Clinical Trials ◽

Phase I ◽

Tumor Cells ◽

Neutralizing Antibodies ◽

Ebola Virus ◽

Vaccine Development ◽

Rna Viruses ◽

Antibody Responses ◽

Phase Iii ◽

Phase I Clinical Trials

Self-replicating single-stranded RNA viruses such as alphaviruses, flaviviruses, measles viruses, and rhabdoviruses provide efficient delivery and high-level expression of therapeutic genes due to their high capacity of RNA replication. This has contributed to novel approaches for therapeutic applications including vaccine development and gene therapy-based immunotherapy. Numerous studies in animal tumor models have demonstrated that self-replicating RNA viral vectors can generate antibody responses against infectious agents and tumor cells. Moreover, protection against challenges with pathogenic Ebola virus was obtained in primates immunized with alphaviruses and flaviviruses. Similarly, vaccinated animals have been demonstrated to withstand challenges with lethal doses of tumor cells. Furthermore, clinical trials have been conducted for several indications with self-amplifying RNA viruses. In this context, alphaviruses have been subjected to phase I clinical trials for a cytomegalovirus vaccine generating neutralizing antibodies in healthy volunteers, and for antigen delivery to dendritic cells providing clinically relevant antibody responses in cancer patients, respectively. Likewise, rhabdovirus particles have been subjected to phase I/II clinical trials showing good safety and immunogenicity against Ebola virus. Rhabdoviruses have generated promising results in phase III trials against Ebola virus. The purpose of this review is to summarize the achievements of using self-replicating RNA viruses for RNA therapy based on preclinical animal studies and clinical trials in humans.

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Efficacy and Safety of SOBERANA 02, a COVID-19 conjugate vaccine in heterologous three doses combination

10.1101/2021.10.31.21265703 ◽

2021 ◽

Author(s):

Maria Eugenia Toledo-Romani ◽

Mayra Garcia-Carmenate ◽

Carmen Valenzuela Silva ◽

Waldemar Baldoquin-Rodriguez ◽

Marisel Martinez Perez ◽

...

Keyword(s):

Clinical Trials ◽

Placebo Group ◽

Conjugate Vaccine ◽

Neutralizing Antibodies ◽

Phase 1 ◽

Dose Schedule ◽

T Cell Response ◽

Group 14 ◽

Phase 3 ◽

Double Blinded

Background: SOBERANA 02 is a COVID19 conjugate vaccine (recombinant RBD conjugated to tetanus toxoid). Phase 1 and 2 clinical trials demonstrated its high immunogenicity, promoting neutralizing IgG together with specific T-cell response. A third dose of SOBERANA Plus (SARS-CoV-2 RBD-dimer) further increased the specific anti-RBD neutralizing antibodies. Methods: In a randomized, double-blinded, placebo-controlled, phase 3 trial we randomly assigned 44 031 participants, aged 19-80 years to three groups in a 1:1:1 ratio to receive 28 days apart either a) two doses of 25 microg SOBERANA 02, or b) two doses of 25 microg SOBERANA 02 followed by a third dose of 50 microg SOBERANA Plus, or c) two doses of placebo. Reported study endpoints are vaccine efficacy (VE) evaluated through laboratory-confirmed symptomatic COVID-19 cases and safety. During the trial, the SARS CoV-2 isolates in Havana were predominantly (beta 74.0 %) and shift gradually to delta (100%). Results: Two doses of SOBERANA 02 protects against symptomatic COVID-19: 43 cases in the two-dose group (14 371) vs. 155 in the placebo group (14 403), VE 71.0%, adjusted (CI 95%58.9-79.1). The heterologous three dose combination with SOBERANA Plus protected against symptomatic COVID-19: 15 cases in the vaccine groups (13 833) vs. 155 in the placebo group (14 303), VE 92.4%, adjusted (CI 95% 86.9-95.6%). For two-dose schedule VE against severe COVID-19 was 63.0% and for death 59.0%; for heterologous three-dose schedule, 100% in both cases. Conclusions: This is the first phase 3 study of a three-dose, heterologous vaccine combination against SARS-CoV-2. Two doses of the conjugate vaccine SOBERANA 02 was safe and attained efficacy of 71.0% in adults population 19-80 y/o; incorporating SOBERANA Plus after two doses of SOBERANA 02, increased efficacy from 71.0 % to 92.4% (Clinical Trials IFV/COR/09 number, RPCEC00000354.)

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The clinical advances of proteolysis targeting chimeras in oncology

Exploration of Targeted Anti-tumor Therapy ◽

10.37349/etat.2021.00061 ◽

2021 ◽

Author(s):

Hao Xie ◽

Junjia Liu ◽

Diego M. Alem Glison ◽

Jason B. Fleming

Keyword(s):

Clinical Trials ◽

Small Molecules ◽

Phase 1 ◽

Hematologic Malignancies ◽

Clinical Findings ◽

Preclinical Development ◽

Protein Targets ◽

Initial Discovery ◽

Clinical Advances ◽

Initial Clinical Evaluation

Proteolysis targeting chimeras (PROTACs) are a class of small molecules designed to target proteins for degradation. Their novel and unique modes of action provide PROTACs with the potential for their application in the management of both solid and hematologic malignancies. Since its initial discovery, the technology of targeted protein degradation, especially in the form of PROTACs, has had significant advances. A number of PROTACs have entered a late stage of preclinical development. Several of them are either in phase 1/2 clinical trials or approaching approval for initial clinical evaluation. This article discusses the preclinical and clinical findings of PROTACs of clinically relevant protein targets in cancer.

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Regulation of Chemoresistance and Immune Resistance of B-NHL Cell Lines by Overexpression of YY1 and Bcl-xL, Respectively: Reversal of Resistance by Rituximab.

Blood ◽

10.1182/blood.v106.11.1517.1517 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1517-1517

Author(s):

Mario I. Vega ◽

Ali R. Jazirehi ◽

Sara Huerta-Yepez ◽

Benjamin Bonavida

Keyword(s):

Tumor Cells ◽

Cell Lines ◽

Drug Induced ◽

Direct Role ◽

No Donor ◽

Immune Resistance ◽

Apoptosis Inhibition ◽

Immune Mediated ◽

Induced Apoptosis

Abstract We have recently reported that treatment of B-NHL cell lines with rituximab sensitizes the tumor cells to both chemotherapy and Fas-induced apoptosis (Jazirehi and Bonavida, 2005, Oncogene, 24:2121–2145). This study investigated the underlying molecular mechanism of rituximab-mediated reversal of resistance. Treatment of B-NHL cell lines inhibited the constitutively activated NF- κB. Cells expressing dominant active IκB or treated with NF-κB specific inhibitors were sensitized to both drugs and FasL agonist mAb (CH-11)-induced apoptosis. Downregulation of Bcl-xL expression via inhibition of NF-κB activity correlated with chemosensitivity. The direct role of Bcl-xL in chemoresistance was demonstrated by the use of Bcl-xL overexpressing Ramos cells, Ramos HA-BclxL (gift from Genhong Cheng, UCLA), which were not sensitized by rituximab to drug-induced apoptosis. However, inhibition of Bcl-xL in Ramos HA-Bcl-x resulted in sensitization to drug-induced apoptosis. The role of Bcl-xL expression in the regulation of Fas resistance was not apparent as Ramos HA-Bcl cells were as sensitive as the wild type cells to CH-11-induced apoptosis. Several lines of evidence support the direct role of the transcription repressor Yin-Yang 1 (YY1) in the regulation of resistance to CH-11-induced apoptosis. Inhibition of YY1 activity by either rituximab, the NO donor DETANONOate, or following transfection with YY1 siRNA all resulted in upregulation of Fas expression and sensitization to CH-11-induced apoptosis. These findings suggest two complementary mechanisms underlying the chemo-sensitization and immuno-sensitization of B NHL cells by rituximab via inhibition of NF-κB. The regulation of chemoresistance by NF-κB is mediated via Bcl-xL expression whereas the regulation of Fas resistance by NF-κB is mediated via YY1 expression and activity. These findings suggest that drug-resistant NHL tumor cells may be sensitive to immune-mediated therapeutics.

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Mechanism of Obatoclax (GX15-070; Bcl-2 Family Inhibitor)-Induced-Sensitization of B-NHL Cells to TRAIL-Mediated Apoptosis: Inhibition of the NF-κB Survival Pathway and the DR5 Transcription Repressor Yin Yang1 (YY1)

Blood ◽

10.1182/blood.v112.11.4976.4976 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4976-4976

Author(s):

Melisa Martinez- Paniagua ◽

Mario I. Vega ◽

Sara Huerta-Yepez ◽

Bonilla Gonzalez ◽

Vanessa Suarez ◽

...

Keyword(s):

Tumor Cell ◽

Cell Lines ◽

Type I ◽

Gene Products ◽

Direct Role ◽

Apoptotic Gene ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

Anti Cd20 ◽

Novel Therapeutic Strategies

Abstract Patients with B-NHL respond initially to treatment with rituximab (chimeric anti- CD20 monoclonal antibody) in combination with CHOP. However, a subset of patients does not respond or develop refractoriness to further treatments. Therefore, there is an urgent need to develop novel therapeutic strategies to treat unresponsive patients. We have explored the potential therapeutic efficacy of TRAIL though, most tumors and cell lines are resistant to TRAIL-induced apoptosis. Our previous findings and those of others have demonstrated that the overexpression of anti-apoptotic gene products such as Bcl-2, BclXL, and Mcl-1 regulates resistance to TRAIL and thus, inhibition of these gene products reverses resistance. Hence, we hypothesized that treatment of B-NHL cell lines with the Bcl-2 family inhibitor, Obatoclax (GX15-070; Gemin X Pharmaceuticals, Malvern, PA) will result in tumor cell sensitization to TRAIL apoptosis. We have used the B-NHL Ramos cell line as model. Treatment of Ramos cells with various concentrations of Obatoclax (7–28 nM) and TRAIL (2.5–20 ng/ml) resulted in significant potentiation of apoptosis and the combination treatment was synergistic. We then explored the mechanism of Obatoclax-induced sensitization to TRAIL. Treatment of Ramos cells with Obatoclax inhibited NF-κB activity and downstream anti-apoptotic gene products regulated by NF- κB (example Bcl-xl, Mcl-1 and XIAP) as assessed by western. Since Obatoclax inhibited NF-κB activity, we explored its effect on the transcription repressor YY1 and DR5 expression. Treatment of Ramos with Obatoclax significantly inhibited YY1 expression concomitantly with upregulation of total and surface DR5 expression that are regulated by NF-κB. The direct role of YY1 in the regulation of resistance to TRAIL was demonstrated by treatment of Ramos with siRNA YY1. Such treated cells showed upregulation of DR5 expression and sensitization to TRAIL apoptosis. The sensitization by Obatoclax resulted in activation of both Type I and Type II apoptotic pathways when used in combination with TRAIL. These findings establish a novel mechanism of Obatoclax-induced gene modification aside from its direct inhibition of Bcl-2 family. Further, our findings with Obatoclax are different from those recently reported by Song et al., [JBC 2008; July 3 (Epub ahead of print)] demonstrating that ABT-737, a small molecule Bcl-2 inhibitor, potentiated TRAIL-induced apoptosis via activation of NF-κB and NF-κB-induced upregulation of DR5 transcription via NF-κB DNA binding site on the DR5 promoter. It is possible that Obatoclax and ABT-737 mediate their sensitization to TRAIL via distinct mechanisms. In summary, our findings demonstrate the potential therapeutic application of Obatoclax in combination with TRAIL or agonist DR4/DR5 antibodies in the reversal of tumor cell resistance to TRAIL.

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Roscovitine Sensitizes Leukemia and Lymphoma Cells to TRAIL-Induced Apoptosis and Enhances Cell-Mediated Cytotoxicity

Blood ◽

10.1182/blood.v116.21.596.596 ◽

2010 ◽

Vol 116 (21) ◽

pp. 596-596

Author(s):

Jan Molinsky ◽

Marie Markova ◽

Magdalena Klanova ◽

Michal Koc ◽

Lenka Beranova ◽

...

Keyword(s):

Clinical Trials ◽

Cell Lines ◽

Low Dose ◽

Hematologic Malignancies ◽

Additive Effect ◽

Cytotoxic Agents ◽

Potential Contribution ◽

Primary Cells ◽

Trail Receptors ◽

Induced Apoptosis

Abstract Abstract 596 Roscovitine is a selective inhibitor of cyclin-dependent kinases (CDK) and it is under evaluation in several clinical trials in the treatment of diverse cancers. TNF-related apoptosis inducing ligand (TRAIL) is a death ligand important for tumor immunosurveillance with selective antitumor activity and minimal toxicity toward tissues. Soluble TRAIL is also under evaluation in several clinical trials. Unfortunatelly, many cancers are resistant to TRAIL. To circumvent TRAIL resistance, there is effort to combinate TRAIL with other cytotoxic agents. By measuring apoptosis and proliferation, we demonstrated that combination of low dose roscovitine and low dose TRAIL (low dose= up to 30% of apoptotic cells after 24h treatment) is synergistic in 20 of 21 tested hematologic cell lines including TRAIL resistant cell lines. Moreover, this combination was tested on primary cells from 9 patients with hematologic malignancies with synergism in 4 of 8 samples from patients with acute myeloid leukemia (AML) and 1 sample from patient with mantle cell lymphoma. Remaining 4 AML samples showed additive effect. Based on these results, we decided to explore molecular mechanisms responsible for the synergism between roscovitine and TRAIL using TRAIL-resistant K562 cells. Despite decreased mRNA, the surface expression of TRAIL receptors remained unaffected after 24h roscovitine treatment. Immunoprecipitation of death-inducing signaling complex (DISC) revealed distinct proapoptotic changes (enhanced CASP8 and 10, reduced FLIP at 12 and 24h). These proapoptotic changes suggested that roscovitine might synergize with other death ligands acting through the DISC, namely TNF and FASLG. Indeed, roscovitine significantly sensitized diverse cell lines (K562, DOHH2, RAMOS) to TNF or FASLG-induced apoptosis. We subsequently proved that pretreatment of the cells (K562, DOHH2, RAMOS) with roscovitine increased by approx. 20% the level of cell-mediated cytotoxicity (peripheral blood mononuclear cells from a healthy volunteer marked with carboxyfluorescein succinimidyl ester). Thus, proapoptotic changes of the DISC seem to play essential role in mediating roscovitine-induced sensitization to TRAIL. Despite detected alterations of the DISC, we decided to unveil additional potential changes in the protein levels of key apoptotic regulators by western blotting at 1.5, 3, 6, 12 and 24h timepoints. Like Ortiz-Ferron et al. we detected gradual downregulation of MCL1 that peaked at 12h, followed, however, by substantial upregulation at 24h. We proved that even at this point, i.e. at 24h exposure to roscovitine, the cells were sensitized to TRAIL-induced apoptosis. The role of MCL1 in mediating the proapoptotic change thus remains elusive. BCL-XL showed similar kinetics as MCL1. Several proapoptotic proteins were overexpressed (BAK and BAD at 1.5h, and PUMA at 1.5h and 24h). Gene-expression profiling unveiled additional changes that might contribute to sensitization to TRAIL, e.g. upregulation of proapoptotic death inducer-obliterator 1 (DIDO1) and downregulation of antiapoptotic DNA-damage-inducible transcript 4 (DDIT4). In contrast to TRAIL (and the other death ligands) roscovitine showed only additive effect or even antagonism with the tested genotoxic agents (cytarabine, doxorubicin, fludarabine, etoposide, cisplatin) probably due to the inhibition of CDK2 by roscovitine (Yu et al., Yanjun et al.). We demonstrated that combination of roscovitine and TRAIL is synergistic in hematologic cell lines and primary cells. In addition, roscovitine was shown to have potent immunostimulatory effect by increasing cell-mediated cytotoxicity. Based on our results we suggest that roscovitine-induced sensitization to TRAIL-triggered apoptosis was mediated by proapoptotic changes of the DISC with potential contribution of the proapoptotic changes in the protein expression of the apoptotic regulators (MCL1, BCL-XL, PUMA, BAK, BAD). We also suggest that roscovitine-induced increase in cell-mediated cytotoxicity, known to be mediated in part through death ligands, was also a consequence of the proapoptotic alteration of the DISC. Roscovitine, as a single agent, or in combination with TRAIL, might have a role in the experimental treatment of selected hematologic malignancies. Financial Support: LC 06044, MSM 0021620806, MSM 0021620808, GAUK 259211/110709, SVV-2010-254260507, IGA MZ NS/10287-3 Disclosures: No relevant conflicts of interest to declare.

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Targeting An Ancient Retrovirus In Tumor Using Engineered T Cells

Blood ◽

10.1182/blood.v122.21.4206.4206 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4206-4206

Author(s):

Janani Krishnamurthy ◽

Brian Rabinovich ◽

Simon Olivares ◽

Mi Teijuan ◽

Kirsten Switzer ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

Tumor Cells ◽

Treatment Strategy ◽

Conflicts Of Interest ◽

Sleeping Beauty ◽

Normal Organ ◽

Car T Cells ◽

Car T ◽

Env Protein

Abstract Human endogenous retroviruses (HERVs) are ancient viruses forming 8% of human genome. One subset of HERVs, the HERV-K has recently been found to be expressed on tumor cells including melanoma, breast cancer and lymphoma but not on normal body cells. Thus, targeting HERV-K protein as a tumor associated antigen (TAA) may be a potential treatment strategy for tumors that are resistant to conventional therapies. One approach to improve therapeutic outcome is by infusing T cells rendered specific for such TAAs preferentially expressed on tumor cells. Recognition of cell-surface TAAs independent of major histocompatibility complex can be achieved by introducing a chimeric antigen receptor (CAR) on T cells using gene therapy. This approach is currently being used in our clinical trials adoptively transferring CD19-specific CAR+ T cells into patients with B-lineage malignancies. Preliminary analysis of HERV-K env protein expression in 268 melanoma samples and 139 normal organ donor tissues using immunohistochemistry demonstrated antigen expression in tumor cells and absence of expression in normal organ tissues. The scFv region from a mouse monoclonal antibody to target HERV-K env was used to generate a CAR and cloned into Sleeping Beauty (SB) plasmid for stable expression in T cells. HERV-K-specific CAR+T cells were selectively propagated ex vivo on artificial antigen presenting cells (aAPC) using an approach already in our clinical trials. Indeed, after genetic modification of T cells and selection on HERV-K+ aAPC, over 95% of propagated T cells stably expressed the introduced HERV-K-specific CAR and exhibited redirected specificity for HERV-K+ melanoma (Figure 1). Further, the adoptive transfer of HERV-K-specific CAR+T cells killed metastatic melanoma in a mouse xenograph model. While we have chosen melanoma as our tumor model, this study has the potential to be applied to other malignancies, including lymphoma and myeloma due to restricted expression of HERV-K envelope (env) protein on these tumor cells. These data demonstrate that it is feasible to generate T cells expressing a HERV-K-specific CAR using a clinically-appealing approach as a treatment strategy for HERV-K env+ tumors. Disclosures: No relevant conflicts of interest to declare.

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Impaired Humoral Immunity to SARS-CoV-2 Vaccination in Non-Hodgkin Lymphoma and CLL Patients

10.1101/2021.06.02.21257804 ◽

2021 ◽

Author(s):

Catherine Diefenbach ◽

Jessica Caro ◽

Akiko Koide ◽

Michael Grossbard ◽

Judith Goldberg ◽

...

Keyword(s):

Hodgkin Lymphoma ◽

Neutralizing Antibodies ◽

Close Contact ◽

Humoral Response ◽

Hematologic Malignancies ◽

Igg Antibody ◽

Neutralization Capacity ◽

Non Hodgkin Lymphoma ◽

Current Guidance ◽

Anti Cd20

Patients with hematologic malignancies are a high priority for SARS-CoV-2 vaccination, yet the benefit they will derive is uncertain. We investigated the humoral response to vaccination in 53 non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), or CLL patients. Peripheral blood was obtained 2 weeks after first vaccination and 6 weeks after second vaccination for antibody profiling using the multiplex bead-binding assay. Serum IgG, IgA, and IgM antibody levels to the spike specific receptor binding domain (RBD) were evaluated as a measure of response. Subsequently, antibody-positive serum were assayed for neutralization capacity against authentic SARS-CoV-2. Histology was 68% lymphoma and 32% CLL; groups were: patients receiving anti- CD20-based therapy (45%), monitored with disease (28%), receiving BTK inhibitors (19%), or chemotherapy (all HL) (8%). SARS-CoV-2 specific RBD IgG antibody response was decreased across all NHL and CLL groups: 25%, 73%, and 40%, respectively. Antibody IgG titers were significantly reduced (p < 0.001) for CD20 treated and targeted therapy patients, and (p = 0.003) for monitored patients. In 94% of patients evaluated after first and second vaccination, antibody titers did not significantly boost after second vaccination. Only 13% of CD20 treated and 13% of monitored patients generated neutralizing antibodies to SARS-CoV-2 with ICD50s 135 to 1767, and 445 and > 10240. This data has profound implications given the current guidance relaxing masking restrictions and for timing of vaccinations. Unless immunity is confirmed with laboratory testing, these patients should continue to mask, socially distance, and to avoid close contact with non-vaccinated individuals.

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Targeted EZH2 Inhibitors in Diffuse Large B-Cell Lymphoma (DLBCL): Immunohistochemical and Mutational Profiles of Patients May Determine Candidates for Treatment

Blood ◽

10.1182/blood.v124.21.1656.1656 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1656-1656 ◽

Cited By ~ 1

Author(s):

Sydney Dubois ◽

Sylvain Mareschal ◽

Marie Cornic ◽

Jean-Michel Picquenot ◽

Philippe Bertrand ◽

...

Keyword(s):

Clinical Trials ◽

B Cell ◽

Sanger Sequencing ◽

Conflicts Of Interest ◽

Phase 1 ◽

B Cell Lymphoma ◽

Staining Intensity ◽

Driver Mutations ◽

Inhibitor Treatment

Abstract DLBCL is the most common lymphoid malignancy, accounting for 30-40% of all Non Hodgkin Lymphomas. Gene expression profiling has identified two main subtypes: Germinal Center B-Cell like (GCB) and Activated B-Cell like (ABC). EZH2 plays an essential role in epigenetic regulation of DLBCL by specifically mono-, bi- and tri-methylating histone H3 lysine 27 (H3K27me1/-me2/-me3). Recurrent somatic heterozygous gain-of-function mutations of EZH2 have been identified in DLBCL, mostly affecting tyrosine 641 (Y641), inducing increased H3K27me3. Novel EZH2 inhibitors are currently being tested in phase 1 and 2 clinical trials in patients with and without EZH2 Y641 mutations, but no study has examined which patients would most benefit from this treatment. We studied a cohort of 100 patients with DLBCL with available biopsies (96 at diagnosis and 4 at relapse) and developed an immunohistochemical (IHC) assay based on antibodies specifically targeting EZH2, H3K27me3 or H3K27me2. Eighty-six biopsies (12 EZH2 Y641 mutant and 74 WT EZH2) were usable for IHC analysis. Biopsies were scored based on staining intensity and percentage of tumor cells stained, and a me3/me2 score (log of me3 to me2 ratio) was calculated for each patient. Sanger sequencing of EZH2was performed on all patients, GCB/ABC signature was determined by DASL technology based on the expression of 19 genes, and t(14;18) translocation was detected by karyotyping and FISH. The cohort was also extended to 15 patients with EZH2 Y641 mutations and 17 WT EZH2 patients for Next Generation Sequencing (NGS) analysis of a panel of 34 genes involved in lymphomagenesis. Among our cohort, 45 patients were ABC, 50 were GCB, and 5 were unclassified. Sanger sequencing identified 14 patients with EZH2 Y641 mutations (12 GCB, 1 ABC, 1 unclassified). The t(14;18) translocation was more frequent in patients with EZH2 Y641 mutations (9/14, 64%) (p<10-4). Three distinct IHC profiles emerged based on me3/me2 score: a me3-high/me2-low profile (me3/me2 score>0, n=12/86), a me3-low/me2-high profile (me3/me2 score<0, n=41/86) and an intermediate profile (me3/me2 score=0, n=33/86). Patients with EZH2 Y641 mutations mostly exhibit me3/me2 score>0 profiles (n=7/12), whereas patients with WT EZH2 are split between intermediate (n=29/74) and me3/me2 score<0 profiles (n=40/74) (p<10-5). Survival analysis was performed on patients with biopsies at diagnosis treated with Rituximab. ABC subtype is associated with both inferior OS and PFS within our cohort (p=0.03); among ABC patients, low EZH2 IHC expression is associated with superior OS (p=0.035) and PFS (p=0.02). No correlation was found between prognosis and IHC profile. All EZH2 mutations were confirmed by NGS along with their Variable Allele Frequency (VAF). Among GCB EZH2 Y641 mutant patients, a majority of clonal EZH2 mutations (n=11/14) and a minority of subclonal EZH2 mutations (n=3/14) were identified by comparing VAFs of EZH2 and of well-known DLBCL/Follicular Lymphoma driver mutations (including TNFRSF14,CREBBP and MYD88: figure). Among the 86 patients, a tendency toward a correlation between me3/me2 score and EZH2 VAF exists (p=0.09, r=0.51) and of the 5 patients with EZH2 Y641 mutations presenting a me3/me2 score ≤0, 3 exhibit a VAF inferior to the median. Two also present a subclonal EZH2mutation. These findings could potentially explain their unexpected IHC profile, and possibly decrease their response to EZH2 inhibitor treatment. Furthermore, 5 WT EZH2 patients present a me3/me2 score>0; 4 are of the ABC subtype, suggesting an EZH2 mutation bypass in ABC patients. NGS analysis also revealed remarkably similar mutational profiles for 2 of these patients, most notably mutations in PRDM1 and PIM1, potentially responsible for EZH2 upregulation by maintaining B cells in GC reaction and possibly justifying EZH2 inhibitor treatment. EZH2 inhibitors are currently being tested in clinical trials in DLBCL as novel and promising weapons in clinicians’ therapeutic arsenal. This study has shown that IHC and mutational profiles can identify patients most likely to benefit from EZH2 inhibitor treatment by highlighting their in vivo hyper-H3K27me3 status, pinpointing associated activating mutations, and determining EZH2mutation clonality. As such, analyzing these parameters could maximize EZH2 inhibitor benefit and potentially serve to grant access to patients who would otherwise not have been considered. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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An ADCC-Independent Mechanism of Rituximab-Mediated B-NHL Cells Depletion Via Sensitization to Cell Death through the Expression of TRAIL on NK Cells

Blood ◽

10.1182/blood.v116.21.4919.4919 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4919-4919

Author(s):

Mario I. Vega ◽

Sara Huerta-Yepez ◽

Melisa Martinez-Paniagua ◽

Stavroula Baritaki ◽

Benjamin Bonavida

Keyword(s):

B Cell ◽

Cytotoxic Activity ◽

Nk Cells ◽

Tumor Cells ◽

Cell Lines ◽

Fas Ligand ◽

Cytotoxic Cells ◽

Induced Apoptosis ◽

Anti Cd20

Abstract Abstract 4919 Rituximab, a chimeric anti-CD20 mAb, has being used, alone or in combination with chemotherapy, in the treatment of patients with B-NHL and rheumatoid arthritis. It is also being tested clinically in the treatment of other B cell malignancies. The mechanisms by which the antibody depletes the B cells have been shown to be mediated via ADCC, CDC, and apoptosis. In addition, the antibody also signals the cells and modifies various survival pathways and sensitizes the resistant tumor cells to various apoptotic stimuli (Jazirehi and Bonavida, Oncogene 24:2121, 2005). The role of the host innate cytotoxic cells, such as NK cells, in cooperation with rituximab in the depletion of B-NHL cells has been poorly explored. Studies by us and others have reported that rituximab sensitizes resistant B-NHL tumor cells to both Fas ligand and TRAIL-induced apoptosis (Bonavida, Oncogene 26:3629, 2007; Daniel, D. et al., Blood 110:4037, 2007). Since NK cells express on the surface TRAIL, we hypothesized that rituximab may also sensitize the TRAIL-resistant tumor cells to NK-mediated cytotoxicity. Accordingly, we have examined various TRAIL-resistant B-NHL cell lines and used peripheral blood-derived purified human NK cells. Treatment of various B-NHL cell lines with rituximab sensitized the cells to TRAIL-induced apoptosis. The mechanism of TRAIL-induced cytotoxicity was found to be the result of TRAIL-induced inhibition of NF-κB and downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) as well as inhibition of anti-apoptotic gene products such as Bclxl. Treatment of various B-NHL cell lines with rituximab, unlike treatment with control IgG1, resulted in significant cytotoxicity in the presence of purified NK cells. The extent of the cytotoxic activity was a function of the E:T ratios used. We then examined the contribution of TRAIL expressed on the NK cell surface for its role in NK-mediated cytotoxicity of rituximab-pretreated B-NHL cells. We used a neutralizing TRAIL antibody that was added in the reaction mixture and demonstrated that the NK cytotoxic activity was significantly reduced compared to controls. These studies with rituximab were also confirmed with other CD20 mAbs. We are currently examining the sensitization of freshly-derived B-NHL and CLL cells that are treated with rituximab and other anti-CD20 mAbs to NK-mediated cytotoxicity for validation of the findings with cell lines. The present findings suggest that, in vivo, patients who are treated with rituximab may recruit NK and other effector cells to mediate, independently of ADCC, cytotoxicity via the TNF-family ligands (e.g. TNF-α, Fas-L, TRAIL). The studies also suggest that this B cell-depletion mechanism by NK cells may contribute to the mechanism of rituximab- mediated depletion of B-NHL cells in vivo. Noteworthy, the proposed host cytotoxic mechanism may not be functional if the therapeutic treatment consists of the combination of rituximab and immunosuppressive chemotherapeutic drugs that may lead to depletion or inactivation of host cytotoxic cells. Disclosures: No relevant conflicts of interest to declare.

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