Lymphoma Lines Research Articles

Pharmacological Inhibitory Effect of Imetelstat, a Novel Human Telomerase Inhibitor in Diffuse Large B-Cell Lymphoma and Peripheral T-Cell Lymphoma

Telomerase activity is present in some non-Hodgkin lymphomas (NHL), including diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphomas (PTCL), and correlated with proliferation index, suggesting anti-telomerase drugs may be efficacious in some types of NHL (Ely et al. 2000). Imetelstat, a telomerase inhibitor that specifically targets the RNA template of human telomerase, is a potent, first-in-class, competitive inhibitor of telomerase enzymatic activity and has demonstrated clinical activity in myeloid malignancies (Steensma et al. 2021; Mascarenhas et al. 2021). No studies have been conducted to demonstrate the effects of imetelstat in DLBCL and PTCL. In our study, we characterized telomerase activity and telomere length, as well as the in-vitro effects of imetelstat on cell viability and apoptosis of a panel of DLBCL and PTCL cell lines. Six DLBCL cell lines, including germinal B cells (GCB: DOHH2, HT, SUDHL4), non-germinal B cells (HBL-1, TMD8), and one high-grade B cell lymphoma line RC with MYC and BCL2 gene rearrangements (double hit lymphomas; ), and five PTCL cell lines (Karpas 299, H9, MAC2A, SUDHL1, and SUPM2) were tested for imetelstat single agent activity as assessed by change of telomerase activity using ScienCell's Telomerase Activity Quantification qPCR Assay Kit, cell viability by Cell Titer-Glo Luminescent Cell Viability Assay and apoptosis by Annexin V/Propidium Iodide flow assay. Baseline telomerase activity and telomere length were measured for each cell line to correlate with imetelstat activity. In addition, PTCL cell lines were treated with Suberoylanilide hydroxamic acid (SAHA) and imetelstat to determine if the combination had an impact on cell viability over either of the single agents. All DLBCL and PTCL cell lines have higher baseline telomerase activity with Ct 20.6-23.2 cycles than the telomerase-positive cell lysate control with Ct 30.6 cycles used in the assay. The dose and time-dependent effects of imetelstat were examined through cell viability and apoptosis assays. Imetelstat sensitivity was higher in DLBCL than in PTCL cell lines used in this study. Preliminary experiments determined that IC50 3-17.5 μM imetelstat markedly reduced cell viability and induced apoptosis in DLBCL cell lines. Specifically, non-GCB DLBCL cell lines (IC50 = 3-4.1 μM) were more sensitive than the GCB-DLBCL (IC50 = 6-8.2 μM). While the level of effects in cell viability and apoptosis by imetelstat varied in the DLBCL cell lines, the double hit DLBCL cell line RC was the least sensitive to imetelstat (IC50 = 17.5 μM) after treatment for 8 days. In contrast, compared to DLBCL cell lines, imetelstat single-agent activity on cell viability was limited in PTCL cell lines, although a time and dose-dependent reduction of telomerase activity were noted. Additionally, the greater inhibitory effect in DLBCL than in PTCL cell lines by imetelstat treatment may be attributed to higher telomerase activity observed in DLBCL compared to PTCL cell lines. Furthermore, the PTCL cell lines had a ~7.3 fold longer telomere length than DLBCL cell lines, which was associated with less response to imetelstat than DLBCL lines. A combination of SAHA with imetelstat was more effective than a single agent alone, synergistic activity was observed in Karpas 299, while other PTCL lines had an additive effect. Our findings indicate that imetelstat was more sensitive in non-GCB subtypes than in GCB-DLBCL. While in PTCL cell lines, the activity of imetelstat was enhanced when combined with SAHA. In the lymphoma landscape, telomerase may be a therapeutic target for ABC-type DLBCL; the novel telomerase inhibitor Imetelstat may present one of the potential treatment options.

ULK1 Blockade Preferentially Targets Germinal Centre B-Cell - Diffuse Large B-Cell Lymphoma Subtype By Attenuating Autophagy, c-MYC and Inflammation

Diffuse Large B-cell Lymphoma (DLBCL) is a heterogeneous lymphoid malignancy. Gene Expression profiling (GEP) has characterized two distinct subsets: Germinal Centre B-cell (GCB) and Activated B-cell (ABC) DLBCL. Oncogenic PI3K-AKT signaling has been identified in primary DLBCL subtypes[1]. PI3K-AKT is activated in response to the loss of PTEN in GCB-DLBCL. In ABC-DLBCL the activation is associated with CD79B mutations and concomitant MYD88 alterations. The Bruton Tyrosine Kinase (BTK) functions as an intermediary kinase. Ibrutinib (BTK inhibitor) has demonstrated promising capability in ABC-DLBCL unlike GCB tumors, warranting further investigations[2]. PI3K-AKT-mTOR signaling is also a known regulator of macro-autophagy (herein referred to as autophagy). ULK1 complex is critical for the initiation of autophagy. Aberrant signaling in this pathway promotes nutritional adaptation to malignant cells and contributes to drug resistance. We hypothesize autophagy facilitates cytoprotection and contributes to drug resistance including R-CHOP and Ibrutinib in GCB-DLBCL. Dual targeting of the BTK with Ibrutinib and autophagy initiating complex(es) would be a strategic method to impair these oncogenic functions. To determine the presence of increased autophagy related genes (ATGs) and its association to R-CHOP treatment outcome, we analyzed the GEP of patients from the REMODL-B trial (R-CHOP: GCB n=240 and ABC n=121)[3]. Gene set enrichment analysis (GSEA) showed no difference between the DLBCL patients indicating mutual autophagy dependency. Genes associated to worst overall survival (OS) in autophagy initiating complex I/ ULK1 complex (ULK1, RB1CC1/FIP200 and ATG13) and complex II (AMBRA1, PIK3C3/VPS34, PIK3R4/VPS15 and USO1/p115) were identified using Kaplan-Meier curves and overlapped according to their respective complexes. Statistical analysis confirmed ULK1 complex was significantly dominate in response to R-CHOP treatment leading to poor OS (p=0.0403). These findings provide a rational to inhibit autophagy via the ULK1 complex in GCB patients. Alike, multiple GCB (n=7) and ABC (n=5) cell lines showed substantial autophagy dependency using LC3B autophagy marker expression. Ibrutinib treatment enhanced autophagic flux in GCB cell lines. Addition of ULK1 complex inhibitor (MRT68921) augmented the anti-tumor activity of Ibrutinib in HT, Oci-Ly1, Oci-Ly18 and SUDHL-6 GCB cell lines and activated caspase dependent apoptosis. Interestingly, ULK1 complex inhibitors (MRT68921/ ULK1-101) induced acute NF-ҡB activation in multiple GCB and ABC lymphoma lines irrespective of their NF-ҡB mutational status. RNA sequencing of GCB and ABC cell lines treated with MRT68921, or vehicle reported diverse ULK1 inhibitory signaling pathways. Oci-Ly1 (GCB, c-MYC amplification) treated cells significantly downregulated genes associated to PI3K-mTOR activity/ signaling, autophagy, metabolism, and IL-6. GSEA reported a substantial decrease in c-MYC transcriptional activation/ pathway and TNF signaling. Immunoblotting data confirmed ULK1 inhibition dephosphorylated c-myc(ser62) in a time dependent manner in Oci-Ly1 and Oci-Ly18. In contrast, Oci-Ly3 (ABC, c-MYC amplification) treated cells upregulated genes involved in JAK-STAT pathway, IL-6, and apoptosis. C-MYC remained unaffected. In summary, GCB-DLBCL patients treated with R-CHOP harbor increased autophagy signatures that potentially dictate treatment outcome. Our in vitro data demonstrates ULK1 inhibition sensitizes GCB cell lines to Ibrutinib due to diverse role in regulating NF-ҡB, c-MYC and inflammation. Unlike, ABC, where ULK1 blockade promotes immunogenic-dependent cell death.

High Efficacy of the PARP Inhibitor Olaparib Combined with High-Dose Chemotherapy and Autologous Stem-Cell Transplant for Refractory Lymphomas

More active HDC are needed for ASCT for refractory/poor-risk relapsed lymphomas. Enhancement of the activity of HDC through combination with DNA damage repair (DDR) inhibitors is a promising but unexplored avenue. The enzyme poly(ADP-ribose) polymerase (PARP) catalyzes PARylation at the DNA damage site, which initiates DDR. Olaparib, the first FDA-approved PARP inhibitor for use in selected solid tumors, has shown synergy with conventional-dose chemotherapy, albeit with increased myelotoxicity. We studied in vitro the interaction of olaparib, epigenetically potentiated with the histone deacetylase (HDAC) inhibitor vorinostat, with a combination of gemcitabine/busulfan/melphalan (GemBuMel), which we had developed previously. We observed a markedly increased cytotoxicity of vorinostat/GemBuMel when combined with olaparib in refractory B- and T-cell lymphoma lines (Valdez et al., Leuk Lymphoma 2017;58:2705-16). Our preclinical work led us to design and clinically study olaparib plus vorinostat/GemBuMel with ASCT in refractory lymphomas. PATIENTS AND METHODS: Patients ages 15-65 with refractory lymphomas and adequate end-organ function were eligible for this phase I trial (NCT03259503). Olaparib was given from day -11 to -3, at 7 dose levels (DL1-DL7), ranging from 25 to 300 mg PO twice daily. Vorinostat was given at 1,000 mg PO daily (days -10 to -3). Gemcitabine was administered at 2,475 mg/m2/day IV (days -8 and -3), infused as a loading dose of 75 mg/m2 followed by continuous infusion at 10 mg/m2/min over 4 hours. Busulfan dosing targeted 4,000 μM.min-1/day IV (days -8 to -5). Melphalan was infused at 60 mg/m2/day IV (days -3 and -2). Patients with CD20+ tumors received rituximab (375 mg/m2) IV on day -10. ASCT was on day 0. The trial goals were to identify the recommended phase 2 dose (RP2D) of olaparib, and estimate the overall (ORR), and complete response (CR) rates, event-free (EFS) and overall survival (OS) rates of the regimen. RESULTS: Between 10/19 and 06/22 we enrolled 30 patients: 18 Hodgkin, 6 DLBCL and 6 T-NHL, median age 34 (20-61), median prior lines of therapy 3 (range, 2-7), 8 prior CAR-T or other cellular immunotherapies. Eleven patients had PET+ tumors at HDC (3 of them in PD). An olaparib dose of 150 mg PO BID (DL4) was identified as the RP2D. There was 1 TRM (sepsis) at DL5. The toxicity profile was similar to that of vorinostat/GemBuMel without olaparib and included mucositis (17 grade 3, 11 grade 2), asymptomatic elevation of transaminases (10 grade 3, 7 grade 2), hyperbilirubinemia (7 grade 3, 2 grade 2, with no cases of VOD/SOS), colitis (2 grade 3, 3 grade 2), diarrhea (3 grade 3, 3 grade 2), and asymptomatic bradycardia during the HDC regimen (6 grade 1). Neutrophils and platelets engrafted promptly. The ORR and CR rates were both 100%. At median follow-up of 16 (range, 2-34) months, the EFS and OS rates are both 90%. There were only two relapses at 4 and 7 months, respectively, both in patients treated at DL1. Olaparib markedly inhibited PAR levels (measured by ELISA in PBMNC), which decreased significantly from baseline to day -5 (and became undetectable in those patients treated at the RP2D), with rapid PARylation recovery on day -2 (around 12 hours after the last dose of olaparib). CONCLUSION: In this first trial combining a PARP inhibitor and an HDAC inhibitor with HDC, olaparib/vorinostat/GemBuMel was safe and highly active in refractory relapsed lymphomas, including post-CAR-T relapses. Olaparib, in combination with HDAC inhibition, strongly potentiates HDC through inhibition of PARylation and DDR during treatment. The clinical data are encouraging and this approach warrants further evaluation.

The proto-oncogene TCL1A deregulates cell cycle and genomic stability in CLL

AbstractUpregulation of the proto-oncogene T-cell leukemia/lymphoma 1A (TCL1A) is causally implicated in various B-cell and T-cell malignancies. High-level TCL1A correlates with aggressive disease features and inferior clinical outcomes. However, the molecular and cell biological consequences of, particularly nuclear, TCL1A are not fully elucidated. We observed here in mouse models of subcellular site-specific TCL1A-induced lymphomagenesis that TCL1A exerts a strong transforming impact via nuclear topography. In proteomic screens of TCL1A-bound molecules in chronic lymphocytic leukemia (CLL) cells and B-cell lymphoma lines, we identified regulators of cell cycle and DNA repair pathways as novel TCL1A interactors, particularly enriched under induced DNA damage and mitosis. By functional mapping and in silico modeling, we specifically identified the mitotic checkpoint protein, cell division cycle 20 (CDC20), as a direct TCL1A interactor. According to the regulatory impact of TCL1A on the activity of the CDC20-containing mitotic checkpoint and anaphase-promoting complexes during mitotic progression, TCL1A overexpression accelerated cell cycle transition in B-cell lymphoma lines, impaired apoptotic damage responses in association with pronounced chromosome missegregation, and caused cellular aneuploidy in Eμ-TCL1A mice. Among hematopoietic cancers, CDC20 levels seem particularly low in CLL. CDC20 expression negatively correlated with TCL1A and lower expression marked more aggressive and genomically instable disease and cellular phenotypes. Knockdown of Cdc20 in TCL1A-initiated murine CLL promoted aneuploidy and leukemic acceleration. Taken together, we discovered a novel cell cycle–associated effect of TCL1A abrogating controlled cell cycle transition. This adds to our concept of oncogenic TCL1A by targeting genome stability. Overall, we propose that TCL1A acts as a pleiotropic adapter molecule with a synergistic net effect of multiple hijacked pathways.

P1291: BTM-3566, A NOVEL ACTIVATOR OF THE MITOCHONDRIAL STRESS RESPONSE ERADICATES DIFFUSE LARGE B-CELL LYMPHOMA IN MICE

Background: Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or after CAR-T-cell therapy. Aims: We describe and characterize BTM-3566, a first-in-class compound that activates the OMA1-dependent mitochondrial stress pathway. BTM-3566 is active against a variety of B-cell malignancies but has the greatest effect in DLBCL, inducing robust therapeutic responses in vitro and in vivo. Methods: Chemical biology, animal models, functional genetics Results: BTM-3566 is a small molecule based on a pyrazolothiazol-backbone. BTM-3566 induces apoptosis and complete cell killing in DLBCL lines with an IC50 of ~300 nM, including ABC, GCB, double-hit and triple-hit lymphoma lines. BTM-3566 has > 50% of oral bioavailability and 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. In a dose-finding study using the DLBCL line SUDHL-10, once daily oral treatment with 20 mg/kg BTM-3566 for 21 days resulted in complete regression in all tumor-bearing animals. Importantly, no subsequent tumor growth was observed for 2 weeks after cessation of therapy. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1). Transcriptome analyses revealed that BTM-3566 activates the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and upregulation of the stress transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined that HRI (EIF2AK1) was uniquely required for BTM-3566 eIF2a phosphorylation, activation of the ATF4-ISR and induction of apoptosis. HRI is activated by mitochondrial-related stress, including heme depletion, ROS generation or blockage of mitochondrial ATP synthesis which all result in the activation of the mitochondrial protease OMA1. We found that BTM-3566 activates OMA1 in a manner unrelated to changes in mitochondrial ATP synthesis, reactive oxygen species generation or electron transport chain inhibition. OMA1 activation was required for the therapeutic effect and CRISPR-Cas9 depletion of OMA1 eliminated BTM-3566’s apoptotic activity. Substrates of OMA1 include the mitochondrial GTPase OPA1 and DELE1, a mitochondrial protein recently shown to act as a sensor of mitochondrial dysfunction and to signal through HRI kinase and ATF4. We show that downstream of OMA1 the apoptotic stress signal is relayed to HRI by DELE1, whereas OMA1-dependent OPA1 cleavage and mitochondrial fragmentation were dispensable for the therapeutic effect. Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a poorly characterized mitochondrial membrane protein, negatively correlated with response to BTM-3566. Overexpression of FAM210B prevented OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis and cell cycle arrest. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation. Image:Summary/Conclusion: We describe a novel antitumor mechanism in DLBCL, where BTM-3566 induces mitochondrial stress, activating the OMA1-DELE1-HRI-eIF2a-ATF4 pathway leading to apoptosis and tumor regression. An IND application in DLBCL has been completed with initiation of first in human clinical trials planned in 2022.

Abstract 2655: The GPCR OR13A1 is essential for lymphoma cells

Abstract Background. G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to endogenous (amines, cations, lipids, peptides, glycoproteins) and exogenous (light, tastants, odorants) stimuli. Altered signaling pathways associated with GPCRs are implicated in the pathophysiology of various diseases, such as CXCR3/4/5, S1PR1/2/3, GPR34 in lymphomas. Olfactory receptors (ORs) are GPCRs, initially identified and characterized for their expression in olfactory neurons but now recognized as expressed in different tissues. OR51E1 is known as prostate specific G protein coupled receptor 2 (PSGR2) due to its expression in prostate cancer, OR2B6 and OR2W3 are expressed in breast cancer, and OR51E1 in melanoma. OR13A1 has been included in the double-hit expression signature reported in diffuse large B cell lymphoma (DLBCL) (Ennishi et al 2019). Here, we studied OR13A1 in lymphoma cell lines and clinical specimens. Methods. RNA-Seq and real-time PCR for evaluating the expression of ORs in DLBCL cell lines. Gene expression data for clinical samples obtained from public dataset phs001444.v2.p1. OR13A1 silencing performed with both siRNAs (OCI-Ly1, U2932) and doxycycline-inducible shRNAs (U2932); viability assessed after 3 days using MTT and for longer with Incucyte live imaging. Computer modelling performed based on available ligands for the murine orthologue Olfr211 and similar known GPCRs structure. Results. Only one OR per cell line was usually expressed, as reported for olfactory cells. Using RNA-Seq and real-time PCR, OR13A1 gene appeared the most abundantly expressed in lymphomas cell lines compared to others ORs. Its expression was seen in 35/47 B cell lymphoma lines, with higher expression in germinal center B cell (GCB) DLBCL than activated B cell like (ABC) DLBCL (P 0.03). A similar pattern was observed in patients: higher expression in GCB than ABC and in EZB than MCD (P < 0.001). OR13A1 also appeared higher in normal GC cells than in naïve and memory B cells. Among GCB DLBCL patients, OR13A1 expression was associated with inferior outcome. Silencing of OR13A1 with siRNAs in GCB and ABC DLBCL cells caused > 50% reduction in cell viability, and pERK downregulation. Viability data were confirmed using a doxycycline-inducible shRNA system and live cell imaging instrument. Natural compounds reported as ligands for the murine orthologue Olfr211 or in silico identified to bind as agonists (ex: tyramine) increased cell line growth and compounds predicted to bind as antagonists (ex: cyclohexanone) decreased the cell viability in the presence but not in the absence of the GPCR. Conclusion. OR13A1 gene appeared more abundantly expressed in lymphomas compared to others ORs, higher in GCB than ABC DLBCL and with a possible impact on clinical outcome. Genetic and chemical experiments showed that this GPCR has an essential function in lymphoma cells and could represent a novel therapeutic target. Citation Format: Giulio Sartori, Luciano Cascione, Milos Matkovic, Hiroaki Matsunami, Andrea Cavalli, Francesco Bertoni. The GPCR OR13A1 is essential for lymphoma cells [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 2655.

Comprehensive Comparison of Novel Bovine Leukemia Virus (BLV) Integration Sites between B-Cell Lymphoma Lines BLSC-KU1 and BLSC-KU17 Using the Viral DNA Capture High-Throughput Sequencing Method

Bovine leukemia virus (BLV) infects cattle and integrates into host DNA, causing enzootic bovine leukosis (EBL), an aggressive B-cell lymphoma. Here, we developed a novel proviral DNA-capture sequencing (proviral DNA-capture-seq) method investigating BLV proviral integration in two B-cell lymphoma lines, BLSC-KU1 and BLSC-KU17, derived from BLV-infected cattle with EBL. We designed BLV-specific biotinylated probes to capture the provirus genome and enrich libraries for next-generation sequencing. Validation showed high specificity and efficient enrichment of target sequence reads as well as identification of three BLV proviral integration sites on BLV persistently infected FLK-BLV cells as a positive control. We successfully detected a single BLV proviral integration site on chromosome 19 of BLSC-KU1 and chromosome 9 of BLSC-KU17, which were confirmed by standard PCR and Sanger sequencing. Further, a defective provirus in BLSC-KU1 and complete BLV proviral sequence in BLSC-KU17 were confirmed using long PCR and sequencing. This is the first study to provide comprehensive information on BLV proviral structure and viral integration in BLSC-KU1 and BLSC-KU17. Moreover, the proposed method can facilitate understanding of the detailed mechanisms underlying BLV-induced leukemogenesis and may be used as an innovative tool to screen BLV-infected cattle at risk at an earlier stage than those that have already developed lymphoma.

A First-in-Class STAT3 Degrader KT-333 in Development for Treatment of Hematologic Cancers

Signal Transducer and Activator of Transcription 3 (STAT3) plays important roles in the transduction of signals from growth factors and cytokines in both normal and malignant cells. Upon activation, STAT3 controls expression of genes that regulate cell growth, survival, differentiation, stemness and cell-cell interactions. Aberrant activation of STAT3 has been observed in many cancers including lymphoma and leukemias through activating mutations, hyper-signaling through upstream regulators or loss of negative feedback regulation. Additionally, STAT3-mediated cross-talk in the tumor microenvironment results in suppression of immune surveillance compromising anti-tumor immunity.STAT3 has been historically considered “undruggable”. Heterobifunctional degraders that recruit endogenous E3 ligases to ubiquitinate substrate proteins leading to their degradation by the proteosome have emerged as a novel therapeutic modality with great potentials to drug undrugged protein targets. Here we introduce a first-in-class, potent and selective STAT3 degrader KT-333 that is being developed for the treatment of hematologic malignancies and solid tumors.KT-333 is a small molecule targeted protein degrader of STAT3 and was discovered by an iterative medicinal chemistry SAR campaign improving parameters such as potency, selectivity and ADME profile. It has high solubility in PBS buffer (103 mg/mL, pH7.4). low to moderate clearance in rat, dog and monkey, and moderate plasma half lives across pre-clinical species.KT-333 exhibits potent and selective degradation of STAT3 with DC 50 of 2.5 -11.8 nM in four anaplastic T cell lymphoma (ALCL) lines. Selective degradation of STAT3 over nearly 9000 proteins, including other STAT family members, was demonstrated by mass spectrometry in human PBMCs . Tumor cells expressing NPM-ALK fusion oncoproteins have activated STAT3 and dependency on STAT3 signaling for survival KT-333 treatment led to rapid apoptosis of these ALK+ALCL cells. To better understand the cellular responses to STAT3 degradation, time-course transcriptomic and proteomic analyses were performed using SU-DHL-1 cells treated with KT-333. Time-dependent changes in both mRNA and protein levels of STAT3-regulated genes and pathways were observed: at 8 h only STAT3 showed significant depletion, at 24 h canonical STAT3 downstream genes including SOCS3, IL-2RA and GRZMB showed modulation and at 48 h, pathways including interferon response and cell cycle were significantly enriched. A comparison of the transcriptomic and proteomic data sets showed high correlation. Furthermore, changes in mRNA and protein levels of bona fide STAT3 regulated targets showed a high degree of concordance between SU-DHL-1 cells and SU-DHL-1 xenograft tumors.Preclinical in vitro experiments have demonstrated that approximately 90% degradation of STAT3 for 48 h was sufficient to drive ALK+ ALCL lines into irreversible cell growth inhibition and death. In vivo xenograft studies of SU-DHL-1 and SUP-M2 ALCL models demonstrated dose-dependent tumor growth suppression. An indirect response model that linked exposure to target degradation was established to describe the relationship between exposure and target degradation in the tumor. Consistent with the in vitro data, PK-PD-efficacy analysis in the SU-DHL-1 xenograft model showed that tumor regression could be achieved with intermittent dosing schedules that achieved ~90% degradation of STAT3 for about 48 h over a weekly or bi-weekly dosing interval. . Based on preclinical ADME and PK/PD/efficacy relationship, KT-333 is predicted to drive tumor regression using weekly doses.In conclusion, we have identified a potent STAT3 degrader as development candidate that can be administered parenterally in the clinic on an intermittent dosing regimen that is predicted to be both efficacious and tolerated in STAT3-dependent hematologic malignancies. DisclosuresLiu: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dixit: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Walther: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rong: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yang: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollerkeri: BEAM Therapeutics: Current Employment, Current equity holder in publicly-traded company; Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. DeSavi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Btm-3566, a Novel Activator of the Mitochondrial Stress Response Promotes Robust Therapeutic Responses in Vitro and In Vivo in Diffuse Large B-Cell Lymphoma

Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or patients who fail CAR-T-cell therapy. r/r-DLBCLs are highly heterogeneous both clinically and molecularly, which imposes a pressing need to develop novel therapies to improve outcomes in patients independently of the molecular subtype. We describe here BTM-3566, a first-in-class compound with activity against a variety of B-cell malignancies but with greatest effect in DLBCL. BTM-3566 activates the mitochondrial integrated stress response (ISR) through a novel mechanism regulated by the mitochondrial protein FAM210B. BTM-3566 induces apoptosis in DLBCL lines in vitro and complete tumor regression in vivo in DLBCL PDX mouse models harboring genetic alterations associated with poor prognosis.BTM-3566 is an oral small molecule based on a pyrazolothiazol-backbone, developed for treatment of diffuse large B-cell lymphoma (DLBCL). BTM-3566 induces apoptosis and complete cell killing in DLBCL lines a with an IC 50 of ~200 - 500 nM. Responsive DLBCL cell lines include ABC, GCB, and double-hit and triple-hit lymphoma lines. Pharmacokinetic studies in mice showed suitability for once daily dosing, with > 50% of oral bioavailability and close to 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. BTM-3566 showed stability in human hepatocytes (IC < 5 ml/min*kg) as well and a favorable in vitro safety profile. In xenograft models using the double-hit DLBCL line SU-DHL-10, BTM-3566 treatment resulted in complete regression in all tumor-bearing animals. Most importantly, no subsequent tumor growth occurred for 2 weeks after cessation of therapy, indicating that treatment with BTM-3566 resulted in a durable complete remission in this model of double-hit DLBCL. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, including Myd88 mutations and MYC and BCL2 rearrangements, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1).Transcriptome and proteome analyses revealed that BTM-3566 strongly activated the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and subsequent upregulation of the transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined, using CRISPR-Cas 9 gene depletion, that HRI was uniquely required for BTM-3566 eIF2a phosphorylation, induction of ATF4 ISR and apoptosis. HRI is described as being activated by mitochondrial-related stress, including heme depletion, increased ROS generation or blockage of mitochondrial ATP synthesis which result in an increase in mitochondrial proteostasis including activation of mitochondrial protease OMA1. We determined that BTM-3566 activates OMA1 without acting as a classical mitochondrial toxin. Treatment with BTM-3566 induced OMA1-dependent OPA1 processing and mitochondrial fragmentation in as little as 30 minutes of treatment, in the absence of any reduction in mitochondrial oxygen consumption or membrane depolarization. This data indicates that BTM-3566 represents a new class of compounds that activate the mitochondrial protease OMA1.Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a mitochondrial membrane protein, negatively correlated with response to BTM-3566. Notably, overexpression of FAM210B completely prevents OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis in DLBCL cell line BJAB and the Burkitt lymphoma cell line Ramos. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation.In summary, we describe here a novel, highly potent activator of the mitochondrial ISR, which is well tolerated in mice and dogs, has favorable pharmacokinetics and induces robust DLBCL regression in-vivo. An IND application in B-cell malignancies will be completed by early Q1 2022 with initiation of first in human clinical trials the first half of 2022. [Display omitted] DisclosuresSchwarzer: Bantam Pharmaceutical: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anantha: Bantam Pharmaceutical: Consultancy, Current Employment, Current holder of stock options in a privately-held company. Cooper: Bantam Pharmaceutical: Consultancy. Hannink: Bantam Pharmaceutical: Research Funding. Hembrough: Bantam Pharmaceutical: Consultancy. Levine: Bantam Pharmaceutical: Consultancy, Current holder of individual stocks in a privately-held company. Luther: Bantam Pharmaceutical: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months, Membership on an entity's Board of Directors or advisory committees. Stocum: Bantam Pharmaceutical: Current Employment, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Liesa-Roig: Bantam Pharmaceutical: Research Funding. Kostura: Bantam Pharmaceutical: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties: Named Inventor on patents with assignment to Bantam Pharmaceutical.

MCIR1: A patient‐derived mantle cell lymphoma line for discovering new treatments for ibrutinib resistance

Despite the unprecedented success of ibrutinib in lymphoma therapy, the development of ibrutinib resistance due to acquired BTK or PLCγ2 mutations has become a new clinical problem. However, not all resistance is mediated by these mutations and these mechanisms are poorly understood due to a lack of study tools that truly recapitulate this clinical scenario. We established a novel patient-derived ibrutinib-resistant mantle cell lymphoma (MCL) line named MCIR1. Using immunological, molecular, and cytogenetic approaches, we comprehensively characterized MCIR1 and further demonstrated its utility in the study of resistance mechanisms and treatments to overcome this resistance. We show that MCIR1 is a bona fide ibrutinib-resistant MCL cell line with normal BTK-/PLCγ2 but ibrutinib-resistant ERK1/2 and AKT1 signaling. RNA-Seq analysis revealed a robust non-canonical NF-kB signaling that drives the ibrutinib resistance. We also demonstrate the potential utility of a MCIR1-based cell and mouse model for the discovery of new treatments to overcome BTK inhibitor resistance. We have established the first patient-derived ibrutinib-resistant MCL cell line MCIR1 that lacks BTK or PLCγ2 mutations but exhibits a hyperactive non-canonical NF-kB pathway. We further demonstrate its utility in the discovery and validation of new drugs to overcome this resistance.

GSK137, a potent small-molecule BCL6 inhibitor with in vivo activity, suppresses antibody responses in mice

B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB–POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB–POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB–POZ domain. The compound has good solubility (128 μg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.

Sy-1365, a Potent and Selective CDK7 Inhibitor, Exhibits Anti-Tumor Activity in Preclinical Models of Hematologic Malignancies, and Demonstrates Interactions with the BCL-XL/BCL2 Mitochondrial Apoptosis Signaling Pathway in Leukemia

Introduction: The transcriptional kinase CDK7 has been implicated in the pathogenesis of multiple malignancies, including hematologic malignancies, and may play important roles in regulation of oncogenic transcriptional dependencies and in regulation of the mitochondrial apoptosis machinery in tumors. SY-1365, a selective inhibitor of CDK7, was developed to exploit tumor dependencies driven by CDK7 and is in clinical development in patients with advanced solid tumors. We have previously reported that SY-1365 selectively induces apoptosis in leukemic cells relative to non-malignant cells in vitro, and demonstrates anti-tumor activity in AML xenografts. Here we report that SY-1365 has potent inhibitory activity against a broad panel of hematologic malignancies in vitro and ex vivo, induces regressions in AML xenografts using a clinically relevant dosing regimen, and demonstrates synergy with the BCL2 inhibitor venetoclax in AML cells in vitro.Methods: SY-1365 dose-response curves were measured using the ATP-lite assay in a panel of 85 leukemia and lymphoma cell lines in vitro, and in a panel of 226 patient-derived tumor samples from 5 leukemic indications cultured ex vivo. In vivo tumor inhibitory activity was evaluated using once weekly (QW) and twice weekly (BIW) dosing regimens in ML-2 and Kasumi-1 AML xenografts. Growth-rate (GR) adjusted dose response curves were used to classify cell lines into low and high sensitivity groups, and were evaluated relative to RNA expression data to identify markers predictive of sensitivity to SY-1365. Synergy between SY-1365 and venetoclax was evaluated in vitro using KG-1 AML cells.Results: In 42 leukemia lines, SY-1365 had a median IC50 of 11nM (range 1-79nM), with 48% (20/42) demonstrating particularly low IC50s (<10nM). In 43 lymphoma lines, the median IC50 was 6nM (range 1-39nM), with 59% (27/46) demonstrating low IC50s (<10nM). Similarly, SY-1365 demonstrated potent inhibitory activity against patient-derived leukemia samples cultured ex vivo (median IC50 of 28nM), with 82% (185/226) of the samples showing IC50 below 100nM and 8% (19/226) under 10nM. Furthermore, analysis of growth rate (GR) in hematologic tumor cell lines showed that <10nM SY-1365 induced cytotoxicity (GR<0) in 19% (8/42) of leukemia and 21% (9/43) of lymphoma lines. To evaluate SY-1365 anti-tumor activity in vivo, 2 AML cell lines that demonstrate cytotoxic responses to SY-1365 in vitro (Kasumi-1 and ML-2) were grown as subcutaneous xenografts in mice. QW and BIW dosing of 40mg/kg SY-1365 induced tumor regressions in Kasumi-1 xenografts, and BIW dosing induced tumor stasis in ML-2 xenografts. Tumor growth inhibition was detected with a 20mg/kg BIW dosing regimen in both models. Comparison of SY-1365 sensitivity with RNA expression in hematologic tumor cell lines revealed that lower expression of BCL2L1, which encodes the mitochondrial apoptosis inhibitor BCL-XL, was highly predictive of sensitivity in leukemia cell lines (Accuracy=89%, FDR<0.05); no such relationship was observed in lymphoma lines. Considering the established role of BCL2 and the mitochondrial apoptosis pathway in leukemia, we reasoned that BCL-XL low-expressing leukemias, in addition to being sensitive to SY-1365, would also be reliant on BCL2 for survival. Indeed, in vitro combination studies demonstrated synergistic inhibitory activity of SY-1365 with the BCL2 inhibitor venetoclax in KG-1 AML cells. A potential synergistic interaction with SY-1365 and venetoclax is currently being explored in vivo with AML xenografts.Conclusions: SY-1365 shows potent in vitro inhibitory activity in multiple hematological indications and can induce AML xenograft tumor regression in mice. In vitro exploratory biomarker and tumor cell inhibitory combination studies in AML suggest a role for mitochondrial apoptosis signaling in mediating sensitivity to SY-1365 and support the evaluation of a combination with venetoclax. SY-1365 is currently being assessed in a phase 1 trial in adult patients with advanced solid tumors (NCT03134638). The results described here support the potential for additional exploration in patients with hematological malignancies. DisclosuresHodgson:Syros Pharmaceuticals: Employment, Equity Ownership. Johannessen:Syros Pharmaceuticals: Employment, Equity Ownership. Rajagopal:Syros Pharmaceuticals: Employment, Equity Ownership. Hu:Syros Pharmaceuticals: Employment, Equity Ownership. Orlando:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. McKeown:Syros Pharmaceuticals: Employment. Tyner:Takeda Pharmaceutical Company: Research Funding; Agios Pharmaceuticals: Research Funding; Incyte Corporation: Research Funding; Gilead: Research Funding; Janssen Pharmaceutica: Research Funding; Syros: Research Funding; Aptose Biosciences: Research Funding; Seattle Genetics: Research Funding; Constellation Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; AstraZeneca: Research Funding; Leap Oncology: Consultancy. Sprott:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership. Di Tomaso:Syros Pharmaceuticals: Employment.

Development and Characterization of the Novel Orally Bioavailable EZH1/2 Dual Inhibitor DS-3201

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27) and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 function as catalytic subunits of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Many reports suggest that inhibiting PRC2 activity through EZH2 knockdown gives rise to anti-cancer effect against hematological and solid cancers.To date, some EZH2 selective inhibitors have demonstrated anti-cancer potential in preclinical and clinical studies. In spite of this promise, there is concern that the efficacy anticipated with EZH2 selective inhibitors may be compromised due to compensation by EZH1 activity.Herein, we describe the novel, orally bioavailable EZH1/2 dual inhibitor DS-3201. This compound strongly and selectively inhibited the methyltransferase activity of both EZH1 and EZH2 with an IC50 value of 10 nM and 6.0 nM, respectively in cell-free enzymatic assays. This compound also suppressed trimethylation of H3K27 in cells (IC50: 0.55 nM) more potently than EZH2 selective inhibitors.In order to identify the anti-cancer spectrum of DS-3201, we tested its in vitro growth inhibition activity against a panel of cancer cell lines including a variety of hematological cancers. Among the panel, hematological cell lines showed particular sensitivity to DS-3201, with GI50 values less than 100 nM, including 1) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines harboring fusion genes, such as MLL-AF9, MLL-AF4, or AML1-ETO, 2) diffuse large B-cell lymphoma (DLBCL) cell lines having EZH2 gain-of-function (GOF) mutations, 3) most of the peripheral T-cell lymphoma and multiple myeloma cell lines tested. Our data thus shows this compound has broader anti-cancer activity than EZH2 selective inhibitors, especially in hematology cancers. We also evaluated the in vivo anti-tumor efficacy of DS-3201 against KARPAS-422, a DLBCL tumor xenograft with an EZH2 GOF mutation. A 100 mg/kg once daily oral administration of DS-3201 caused nearly complete tumor regression without any body weight loss and 25 mg/kg administration also showed clear tumor growth delay.Based on these results, we initiated clinical studies of DS-3201 for patients with lymphoma (Clinical trial information: NCT02732275) and for those with AML and ALL (NCT03110354). DisclosuresHonma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Kanno:Asubio Pharma Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Employment. Hirasawa:Daiichi Sankyo Co., Ltd.: Employment. Nosaka:Daiichi Sankyo Co., Ltd.: Employment. Shiroishi:Daiichi Sankyo Co., Ltd.: Employment. Takizawa:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Yasumatsu:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Horiuchi:Daiichi Sankyo Co., Ltd.: Employment. Nakao:Daiichi Sankyo Co., Ltd.: Employment. Suzuki:Daiichi Sankyo Co., Ltd.: Employment. Yamasaki:Daiichi Sankyo Co., Ltd.: Employment. Nakajima:Daiichi Sankyo Co., Ltd.: Employment. Hayakawa:Daiichi Sankyo Co., Ltd.: Employment. Yamazaki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo: Employment. Fujiwara:Daiichi Sankyo Co., Ltd.: Employment.

Repurposing Dasatinib for Ibrutinib-Resistant Diffuse Large B-Cell Lymphoma

Diffuse Large B-Cell Lymphoma (DLBCL) is the most frequent B-cell Non-Hodgkin Lymphoma (B-NHL). It derives from germinal-center (GC) B cells, and has been classified into GC B cell like (GCB) and activated B cell like (ABC) subtypes, which differ in their cell of origin, genetic alterations and responses to therapy, with ABC having inferior prognosis in response to the current immuno-chemotherapeutic regimen (R-CHOP). This standard first-line treatment is successful in ~70% of cases based on 5-year overall survival, but the remaining ~30% of patients remains incurable. While recently developed drugs, including the Bruton Tyrosine Kinase (BTK) inhibitor Ibrutinib and PI3K-delta inhibitors, are providing novel targeted strategies, their impact on DLBCL is still limited to a fraction of cases, mostly in the ABC subtype. In order to identify drugs that can be repositioned for selective efficacy against DLBCL subtypes (GCB- and ABC-DLBCL) compared to non-GC derived lymphomas, we screened a focused set including 2,160 FDA-approved drugs and other targeted drugs in advanced clinical testing on a panel of 9 cell lines consisting of 6 DLBCL (3 ABC and 3 GCB) and 3 Mantle Cell Lymphoma (MCL) lines. This screen identified the multi-kinase inhibitor Dasatinib as the most DLBCL-specific agent. Dose-response experiments on an extended panel of 32 DLBCL lines showed that Dasatinib has broader activity than Ibrutinib, since it is active against all ABC-DLBCL Ibrutinib-sensitive lines, as well as an additional set of 9 ABC- and GCB-DLBCL lines. Notably, Dasatinib overcomes Ibrutinib-resistance caused by BTK C481S mutation, as shown by the fact that Dasatinib is active against 3 Ibrutinib-sensitive ABC-DLBCL lines rendered Ibrutinib-resistant by transduction of BTK C481S mutant. In these cell lines, Dasatinib, analogous to Ibrutinib, is unable to suppress BTK-C481S auto-phosphorylation at Tyrosine 223, indicating that it may act independently of BTK. To understand which kinase or combination of kinases needs to be suppressed for Dasatinib activity, we employed Dasatinib-resistant, gatekeeper mutants of each BCR-associated Src-Family Kinase (LYN, FYN and BLK), which can all be targeted by Dasatinib, alone or in combination. Our results showed that FYN suppression is essential for Dasatinib activity in both DLBCL subtypes. Integrated genomic and bioinformatics approaches to explore genetic determinants of resistance to Dasatinib revealed PTEN disruption as the most significant predictor. PTEN is inactivated in the majority of resistant lines by both genetic and epigenetic mechanisms, resulting in the inability by Dasatinib to restrict PI3K-mediated AKT activation. These data were further validated by showing that 3 Dasatinib-sensitive DLBCL lines were made resistant by transduction of a constitutively active PIK3CA mutant (PIK3CA-H1047R). Notably, suppression of AKT activation by mTORC2 inhibitors can revert Dasatinib resistance, suggesting that combinatorial strategies involving Dasatinib and drugs targeting the PI3K/AKT/mTOR axis may be effective in PTEN-negative DLBCLs. The above results were validated in vivo using DLBCL xenograft mouse models. Overall, these results provide a preclinical framework for Dasatinib-based targeted therapies for DLBCL. DisclosuresNo relevant conflicts of interest to declare.

A Phase 1/2 Study of Oral 5-Azacitidine and Romidepsin in Patients with Lymphoid Malignancies Reveals Promising Activity in Heavily Pretreated Peripheral T-Cell Lymphoma (PTCL)

Introduction: Patients with PTCL are characterized by frequent alterations of epigenetic operations, including recurrent mutations in important genes governing genome wide methylation (i.e., TET2, IDH1/2, and DNMT3). This feature creates the rationale for the use of hypomethylating agents (HMA) in these diseases. Indeed, a recent study showed impressive preliminary activity of 5-azacitidine (AZA) in patients with angioimmunoblastic T-cell lymphoma (AITL). Histone deacetylases (HDACs), transcriptional modulators that operate through modification of histone proteins, are also overexpressed in select types of PTCL. HDAC inhibitors (HDACi), such as romidepsin (ROMI), have shown consistent class activity in patients with R/R PTCL, and received regulatory approval for this indication. Our group has previously shown that the combination of the HDACi and the HMA can ‘reverse’ the malignant signature of T-cell lymphoma lines in vitro. In addition, the combination induced the expression of a unique set of genes not induced by either single agent, including a unique methylation pattern. We therefore launched a phase 1/2 trial of combined AZA and ROMI in patients with lymphoid neoplasms, including PTCL. Herein we present updated analysis of the results of the phase 1 and 2 portions of the trial combined.Methods: Patients with any from of lymphoma were eligible for the phase 1, while the phase 2 exclusively enrolled patients with PTCL. Patients with any number of prior therapies, including transplantation, were eligible for enrollment. In the phase 2 patients with newly diagnosed PTCL were also allowed to participate. Other inclusion criteria were ECOG PS <2 and adequate organ and marrow function. The phase 1 study followed a 3 + 3 dose-escalation design. The starting doses were AZA 100 mg/day and ROMI 10 mg/m2 with progressively increasing dose intensity in subsequent cohorts (7 cohorts). The primary objectives of the study were the maximum tolerated dose (MTD) and dose limiting toxicity (DLT) of the combination. Secondary objectives included ORR (CR + PR), progression free survival (PFS), and duration of response (DOR).Results: 30 patients were enrolled in total, 27 in phase 1 (26 accrued, 1 screening failure) and 3 in phase 2, of whom one has been restaged. In the intention-to-treat population the median age was 49 years (23-79), male-to-female ratio 17:13, and median number of prior therapies 5 (0-18). Sixteen patients had had autologous stem cell transplant, and 1 prior allogeneic transplant. Lymphoma histology's included Hodgkin/other (12, 40%), B-cell (8, 27%), and T-cell (10, 33%). Twenty-seven patients are evaluable for toxicity. We recorded 5 DLTs across all cohorts, 3 of which occurred at the highest dose level (i.e., AZA 300 mg days 1-21 and ROMI 14 mg/m2 days 8, 15, and 22) and included neutropenia (N = 2) and thrombocytopenia (N = 1). Two additional DLTs were 1 pleural effusion and 1 missed dose. The MTD for the combination was AZA 300 mg days 1-14 and ROMI 14 mg/m2 days 8, 15, and 22. Other grade 3-4 toxicities included febrile neutropenia (N = 3), and thrombocytopenia (N = 5), only 2 being grade 4. Among 25 patients evaluable for efficacy (all NHL subtypes), the ORR was 28% (7/25) including a 16% (4/25) CR. Substantially higher response rates were seen in patients with PTCL, where the ORR and CR were 83% (5/6) and 50% (3/6). First dose PK revealed the AUC for R 10 and 14 mg/m2 were 2021.5 ± 1461.3 h*ng/mL, and 1765.9 ± 1002.0 h*ng/mL, respectively. The median t1/2 was 4.8 and 2.4 h, respectively. These data are comparable to historical single agent values.Conclusions: Albeit in a small population, these promising clinical results suggest that a doublet of epigenetic modifiers is well tolerated and may represent a viable backbone to build the next generation of active regimens in PTCL. The complete response seen in the AITL patient enrolled in the phase 2 portion of the study is encouraging and is being specifically expanded. The study is now multicenter, and results on additional patients enrolled in the phase 2 portion will be presented at the meeting. This study is registered on www.clinicaltrials.gov (NCT01998035). DisclosuresO'Connor:Trillium Therapeutics: Research Funding; Celgene: Honoraria, Research Funding.

The Marine Natural Compound Elatol Is a Novel Protein Translation Inhibitor with Potent Activity Against Aggressive Non-Hodgkin Lymphomas

Prognosis remains poor for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and other aggressive non-Hodgkin lymphomas. Targeted small-molecule signaling inhibitors have been poorly active in this setting due to a heterogeneous pathogenesis in which multiple different but overlapping signals may drive lymphomagenesis. Activation of cap-dependent protein translation is a convergence point of multiple lymphoma drivers, including B-cell receptor (BCR) signaling pathways. Cap-dependent protein translation begins with formation of the eIF4F complex, a heterotrimeric complex comprised of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the ATP-hydrolyzing RNA helicase eIF4A, which is required for expression of many oncogenes, including the common lymphoma driver MYC. Despite evidence that disruption of eIF4F function is toxic to lymphoma cell lines and sensitizes mouse models of lymphoma to chemotherapy, eIF4A inhibitors have not progressed to clinical evaluation. We designed a screen to identify novel eIF4A inhibitors by measuring ATP hydrolysis and screened a collection of ~1,200 natural compounds and purified extracts. The marine natural product elatol was the top hit in our screen, showing selectivity for eIF4A over other DEAD-box family helicases and a panel of 97 common kinases. Elatol inhibits eIF4A's helicase activity in cell-free in vitro assays and binds to the target at a 2:1 ratio through interaction with two key lysine residues that fold together in its RNA-binding groove. It also induced apoptosis in a broad range of cancer cell lines from the NCI-60 (average LD50= 1.9 μM; LD50<1μM in 33/60) cancer cell line and CCLE panels, but our collection of non-Hodgkin lymphoma lines showed the greatest sensitivity as a group (average LD50= 1.3 μM; 9/12 <1μM; range 213-5749nM). In DLBCL cell lines, Elatol treatment reduced global protein translation measured through polysome profiling and O-propargyl puromycin incorporation as well as cap-dependent translation measured by luciferase reporter and western blotting. Elatol also blocked translational activation downstream of BCR stimulation in primary chronic lymphocytic leukemia (CLL) cells. The effects of elatol are similar to the rocaglate silvestrol, which inhibits eIF4A though through a different mechanism, but elatol requires higher concentrations to achieve these effects. Elatol's reduced in vitro potency, however, is off-set by a maximum tolerated in vivo dose to mice of 65mg/kg, compared to silvestrol's typical dosing at 0.2-05 mg/kg. Elatol significantly reduced the growth of DLBCL tumors in a xenograft model. Mechanistically, elatol and silvestrol both clearly function downstream of mTOR for translational inhibition, but unlike silvestrol, elatol causes strong and rapid induction of the stress response transcription factor ATF4. Assessment of drug activity in murine embryonal fibroblasts lacking ATF4 and those with an unphosphorylatable mutant allele of its upstream activator eIF2α shows elatol's anti-lymphoma activities are not dependent on ATF4 induction. In addition, an inhibitor of the eIF2α kinase PERK and other evidence demonstrate elatol does not produce an unfolded protein response, but the reason for ATF4 induction is not yet clear and additional eIF2α kinases are under investigation. The novel mechanism of inhibition of ATP hydrolysis may be driving these unique observations compared to other eIF4A inhibitors, but unexpected off targets are also under evaluation. In sum we report a new natural inhibitor of protein translation with potent anti-lymphoma activities. Elatol provides a novel starting point for development of eIF4A inhibitors in cancer therapeutics. DisclosuresNo relevant conflicts of interest to declare.

TRAF3 enhances type I interferon receptor (IFNAR) signaling in T cells through modulation of PTPN22

Abstract T cell responses are critical for successful resolution of many infectious and non-infectious challenges. Robust T cell activation and acquisition of effector functions relies on receipt of at least three signals: 1) antigen recognition by the T cell receptor, 2) co-stimulation, and 3) cytokines. TNF receptor-associated factor 3 (TRAF3) is an adapter protein with diverse cell type- and receptor-specific functions, that regulates signals one and two of conventional T cell activation. In this study, we examine the role of TRAF3 in signaling downstream of the potent signal three cytokine, type I interferon (IFN I). Work from our laboratory has shown that in other contexts, TRAF3 regulates cytokine receptor signaling by facilitating association of phosphatases with JAKs at the cytokine receptor. We therefore hypothesize that TRAF3 regulates IFNAR signaling in T cells through affecting recruitment of the phosphatase PTPN22 to the IFNAR complex. We investigate the role of TRAF3 in IFNAR signaling using a TRAF3-deficient human T cell lymphoma line and T cells from a T cell specific TRAF3 knockout mouse (T-Traf3−/−). TRAF3−/− T cells display a marked defect in STAT1 activation, leading to defects in IFN-stimulated gene expression and CXCL10 secretion. We show TRAF3 and PTPN22 association with JAK1 and IFNAR1 upon IFN I stimulation in WT T cells, and increased association of PTPN22 with JAK1 and IFNAR1 in TRAF3−/− T cells. Inhibition of PTPN22 restores IFNAR signaling in TRAF3-deficient cells to near WT levels. Together, these data indicate that TRAF3 modulates IFNAR signaling by modulating PTPN22 association with the IFNAR signaling complex, broadening our understanding of the crucial signals that drive T cell activation and polarization.

Calcium signaling on Jurkat T cells induced by microbeads coated with novel peptide ligands specific to human CD3ε.

CD3ε is expressed on T lymphocytes as a part of the T cell receptor (TCR)-CD3 complex. Together with other CD3 molecules, CD3ε is responsible for the activation of T cells via transducing the event of antigen recognition by the TCR into intracellular signaling cascades. The present study first aims to identify a novel peptide ligand that binds to human CD3ε in a specific manner and to perform an initial evaluation of its biological efficacy on the human T cell line, Jurkat cells. We screened a phage-display peptide library against human CD3ε using a subtractive biopanning process, from which we identified 13 phage clones displaying unique peptide sequences. One dominant phage clone displaying the 7 amino acid sequence of WSLGYTG, which occupied 90% of tested plaques (18 out of 20) after the 5th round of biopanning, demonstrated a superior binding behavior to other clones in the binding assays against recombinant CD3ε on microbeads or Jurkat cells. The synthesized peptide also showed specific binding to Jurkat cells in a dose-dependent manner but not to B cell lymphoma line, 2PK3 cells. Molecular modeling and docking simulation confirmed that the selected peptide ligand in an energetically stable conformation binds to a pocket of CD3ε that is not hidden by either CD3γ or CD3δ. Lastly, magnetic microbeads conjugated with the synthesized peptide ligands showed a weak but specific association with Jurkat cells and induced the calcium flux, a hallmark indication of proximal T cell receptor signaling, which gave rise to an enhancement of IL-2 section and cell proliferation. The novel peptide ligand and its various multivalent forms have a great potential in applications related to T cell biology and T cell immunotherapy.

Discovery of Potent, Highly Selective, and In Vivo Efficacious, Allosteric MALT1 Inhibitors by Iterative Scaffold Morphing.

MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.

CAR-modified memory-like NK cells exhibit potent responses to NK-resistant lymphomas

AbstractNatural killer (NK) cells are a promising cellular immunotherapy for cancer. Cytokine-induced memory-like (ML) NK cells differentiate after activation with interleukin-12 (IL-12), IL-15, and IL-18, exhibit potent antitumor responses, and safely induce complete remissions in patients with leukemia. However, many cancers are not fully recognized via NK cell receptors. Chimeric antigen receptors (CARs) have been used to enhance tumor-specific recognition by effector lymphocytes. We hypothesized that ML differentiation and CAR engineering would result in complementary improvements in NK cell responses against NK-resistant cancers. To test this idea, peripheral blood ML NK cells were modified to express an anti-CD19 CAR (19-CAR-ML), which displayed significantly increased interferon γ production, degranulation, and specific killing against NK-resistant lymphoma lines and primary targets compared with nonspecific control CAR-ML NK cells or conventional CAR NK cells. The 19-CAR and ML responses were synergistic and CAR specific and required immunoreceptor tyrosine-based activation motif signaling. Furthermore, 19-CAR-ML NK cells generated from lymphoma patients exhibited improved responses against their autologous lymphomas. 19-CAR-ML NK cells controlled lymphoma burden in vivo and improved survival in human xenograft models. Thus, CAR engineering of ML NK cells enhanced responses against resistant cancers and warrants further investigation, with the potential to broaden ML NK cell recognition against a variety of NK cell–resistant tumors.