non-Hodgkin Lymphoma Cell Lines Research Articles

Core regions in immunoglobulin heavy chain enhancers essential for survival of non-Hodgkin lymphoma cells are identified by a CRISPR interference screen.

Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eμ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.

Abstract PO-010: Trial in Progress: Phase 1 Study of ARV-393, a PROTAC BCL6 Degrader, in Advanced Non-Hodgkin Lymphoma

Abstract Background: Non-Hodgkin lymphoma (NHL) represents a biologically and clinically diverse group of hematologic malignancies originating from B cells, T cells, and/or natural killer cells, with those of B-cell origin constituting ∼80–85% of all NHL cases. The BCL6 transcription factor is a key oncogenic driver of B-cell lymphomagenesis, and deregulated BCL6 expression is a common feature of diffuse large B-cell lymphoma (DLBCL), the most common type of NHL. BCL6 translocations have been observed in follicular lymphomas, the second most prevalent NHL, with studies suggesting that these translocations may portend a higher risk of transformation into aggressive lymphoma. BCL6 is also a lineage-defining transcription factor of T follicular helper cells, the cell of origin for angioimmunoblastic T-cell lymphoma (AITL). Moreover, BCL6 expression has been identified in biopsies of patients with AITL, and its continued expression was required for tumor growth in a mouse model of AITL, supporting BCL6 as a cancer driver in this type of NHL as well. ARV-393, an orally administered PROteolysis TArgeting Chimera (PROTAC) BCL6 degrader, is a bifunctional small molecule consisting of a BCL6-binding domain attached by a linker to a cereblon E3 ubiquitin ligase–binding domain. ARV-393 induces ubiquitination and subsequent degradation of BCL6 by the proteasome. In preclinical studies, ARV-393 induced rapid and robust degradation (>90%) of BCL6 in NHL cell lines and demonstrated dose-responsive tumor growth inhibition leading to tumor stasis or regression in xenograft models, supporting further investigation in patients with NHL. This multicenter, first-in-human, phase 1 study will evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of ARV-393 in patients with advanced B-cell NHL or AITL. Methods: Eligible patients (aged ≥18 years) have relapsed/refractory mature B-cell NHL and ≥2 prior systemic therapies, or histologically confirmed AITL that has recurred or progressed following institutional standard-of-care therapy. Patients must also have ≥1 measurable lesion at study entry, Eastern Cooperative Oncology Group performance status of 0 or 1, freshly biopsied or archival tumor tissue available, adequate organ function, no active central nervous system involvement, and no prior allogeneic stem cell transplant or solid organ transplantation. Autologous stem cell transplant must not have occurred ≤100 days and previous CAR T-cell therapy ≤60 days prior to cycle 1 day 1 of ARV-393 treatment. ARV-393 will be administered orally in 28-day cycles with dose escalation according to a Bayesian optimal interval design. The primary objectives are to evaluate the safety and tolerability of ARV-393, determine maximum tolerated dose if necessary, and identify the recommended phase 2 dose(s) and dosing schedule. Secondary objectives are to characterize the pharmacokinetic profile of ARV-393 and evaluate its preliminary anti-tumor activity. Enrollment for this study will open in 2024. Citation Format: Paolo Caimi, Scott Huntington, Sean Landrette, Sheryl M Gough, Vivian Dai, Bryan Jackson, Juan Chavez, Sarah Tannenbaum-Dvir, Matthew Matasar. Trial in Progress: Phase 1 Study of ARV-393, a PROTAC BCL6 Degrader, in Advanced Non-Hodgkin Lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-010.

Comparable efficacy of oral bendamustine versus intravenous administration in treating hematologic malignancies.

The purpose of this study was to analyze potential differences in antitumor efficacy and pharmacokinetics between intravenous (IV) bendamustine and a novel orally administered (PO) bendamustine agent that is utilizing the beneficial properties of superstaturated solid dispersions formulated in nanoparticles. Pharmacokinetics of IV versus PO bendamustine were determined by analysis of plasma samples collected from NSG mice treated with either IV or PO bendamustine. Plasma samples were analyzed using liquid chromatography-mass spectrometry following a liquid-liquid extraction to determine peak bendamustine concentration, area under the concentration-time curve, and the half-life in-vivo. In-vitro cytotoxicity of bendamustine against human non-Hodgkin Burkitt's Lymphoma (Raji), multiple myeloma (MM.1s), and B-cell acute lymphoblastic leukemia (RS4;11) cell lines was determined over time using MTS assays. Luciferase-tagged versions of the aforementioned cell lines were used to determine in-vivo bendamustine cytotoxicity of IV versus PO bendamustine at two different doses. Bendamustine at a high dose in-vitro causes cell death. There was no significant difference in antitumor activity between IV and novel PO bendamustine at a physiologically relevant concentration in all three xenograft models. In-vivo pharmacokinetics showed the oral bioavailability of bendamustine in mice to be 51.4%. The novel oral bendamustine agent tested exhibits good oral bioavailability and systemic exposure for in-vivo antitumor efficacy comparable to IV bendamustine. An oral bendamustine formulation offers exciting clinical potential as an additional method of administration for bendamustine and warrants further evaluation in clinical studies.

Effects of Cannabidiol, ∆9-Tetrahydrocannabinol, and WIN 55-212-22 on the Viability of Canine and Human Non-Hodgkin Lymphoma Cell Lines.

In our previous study, we demonstrated the impact of overexpression of CB1 and CB2 cannabinoid receptors and the inhibitory effect of endocannabinoids (2-arachidonoylglycerol (2-AG) and Anandamide (AEA)) on canine (Canis lupus familiaris) and human (Homo sapiens) non-Hodgkin lymphoma (NHL) cell lines' viability compared to cells treated with a vehicle. The purpose of this study was to demonstrate the anti-cancer effects of the phytocannabinoids, cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), and the synthetic cannabinoid WIN 55-212-22 (WIN) in canine and human lymphoma cell lines and to compare their inhibitory effect to that of endocannabinoids. We used malignant canine B-cell lymphoma (BCL) (1771 and CLB-L1) and T-cell lymphoma (TCL) (CL-1) cell lines, and human BCL cell line (RAMOS). Our cell viability assay results demonstrated, compared to the controls, a biphasic effect (concentration range from 0.5 μM to 50 μM) with a significant reduction in cancer viability for both phytocannabinoids and the synthetic cannabinoid. However, the decrease in cell viability in the TCL CL-1 line was limited to CBD. The results of the biochemical analysis using the 1771 BCL cell line revealed a significant increase in markers of oxidative stress, inflammation, and apoptosis, and a decrease in markers of mitochondrial function in cells treated with the exogenous cannabinoids compared to the control. Based on the IC50 values, CBD was the most potent phytocannabinoid in reducing lymphoma cell viability in 1771, Ramos, and CL-1. Previously, we demonstrated the endocannabinoid AEA to be more potent than 2-AG. Our study suggests that future studies should use CBD and AEA for further cannabinoid testing as they might reduce tumor burden in malignant NHL of canines and humans.

Abstract LB157: Discovery of HP537, a potent and selective p300/CBP inhibitor for the treatment of hematologic malignancies

Abstract E1A-binding protein P300 (p300) and its paralog CREB binding protein (CBP) are key acetyl transferases (HAT) and transcriptional co-factors. p300/CBP bromodomain (BRD) inhibition reduces acetylation of H3K27 at enhancers, leading to suppression of enhancer-driven gene expression, such as MYC and IRF4 that are critical for the initiation, progression, and chemoresistance of hematological malignancies. Thus, p300/CBP inhibition is a highly potential therapeutic strategy for the treatment of hematological malignancies, including multiple myeloma (MM), non-Hodgkin lymphoma (NHL) and leukemia. We have discovered HP537 that competitively inhibits an acetylated peptide substrate to bind BRD of p300/CBP with IC50 < 5 nM. HP537 does not inhibit the enzymatic activity of HAT domain of p300/CBP and HAT1, nor has binding affinity for other BRD containing proteins including BRD4 and BRD9. HP537 inhibits proliferation of a wide range of cell lines derived from MM, leukemia and lymphoma with IC50 values ranged from 0.01 μM to 0.4 μM. Protein expression levels of acetylated H3K27, c-Myc and IRF4 are significantly decreased by HP537 in MM, NHL and acute myelogenous leukemia (AML) cell lines in a dose- and time-dependent manner. HP537 robustly and dose-dependently inhibits tumor growth in OPM-2 (MM), MOLM-16 (AML), and KARPAS-422 (NHL) cell line derived tumor xenograft (CDX) mouse models. Down-regulation of c-Myc and IRF4 are also observed in the tumor samples. In conclusion, HP537 is a novel, potent and selective inhibitor of p300/CBP BRD. The pre-clinical data supports the clinical development of HP537 for the treatment of hematological malignancies. A FIH study of HP537 in cancer patients is planned to start in 2024. HP537 structure will not be disclosed. Citation Format: Jing Li, Zhilin Tu, Jian Peng, Lei Fan, Hua Yu, Fei Wang, Xinghai Li. Discovery of HP537, a potent and selective p300/CBP inhibitor for the treatment of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB157.

Novel CD20 Mutations As a Mechanism of Resistance to CD20-CD3 Targeted Therapies in Non-Hodgkin's Lymphoma

Non-Hodgkin's Lymphoma (NHL) is one of the most common blood cancer types in the world. CD20 is a B cell restricted lineage marker that is a well-established target for the treatment of NHL and anti-CD20 agents are included as in standard of care regimens. Given the success of these targeted therapies, strategies to further enhance therapeutic activity are being explored, including the CD20-CD3 bispecific molecule, mosunetuzumab, which is approved for treatment of relapsed and refractory Follicular Lymphoma in adults who have received >2 lines of treatment. Mosunetuzumab acts by redirecting CD3+ T cells to engage and eliminate CD20-expressing B cells. As with other targeted therapies, mutations and loss of target expression have been described following mosunetuzumab therapy [1]. Here we describe the impact of novel mutations identified in patient biopsy samples from the mosunetuzumab monotherapy trial, GO29781 that were located within the extracellular and transmembrane domains of CD20 on protein expression, localization and T-cell mediated killing. NHL cell lines, SU-DHL-16 and MAVER-1, were engineered using CRISPR-Cas9 system to create CD20 knock out (KO) cell lines. Wild-type (WT) or mutant CD20 were exogenously re-expressed in the CD20 KO cell lines and in the CD20 negative acute lymphoblastic leukemia cell line, REH, to create isogenic cell lines for further analysis. Complete CD20 loss was observed in cells expressing CD20 P160fs (frameshift) and Q187* (truncating) mutations. CD20 missense mutations located in the extracellular domain (C167G, K175E) expressed CD20 protein levels comparable to CD20 WT cells and membrane localization was confirmed by flow cytometry and immunofluorescence microscopy. To test the functional effect of CD20 mutations, in vitro co-culture cell killing assays were carried out using healthy donor CD8+ T cells, isogenic cell lines (3:1 effector to tumor cell ratio) and a proof-of-concept CD20-CD3 bispecific antibody. Differences in T-cell mediated killing were observed across mutation types. CD20 WT cell lines demonstrated 95-99% cell killing following 48 hour treatment while the CD20 KO, frameshift and truncating mutations all showed negligible T-cell mediated killing (~10-17% cell death). Cells expressing the missense mutations in the extracellular domain showed resistance to T-cell mediated killing (~30-45% cell death) compared to CD20 WT cells. T-cell activation was assessed by CD69 (early) and CD25 (late) surface markers. In all co-cultures, the level of CD69+ T cells was equivalent, while the CD69+CD25+ CD8 T cells were only significantly increased in the CD20 WT co-cultures, consistent with the high level of T-cell mediating killing. We have confirmed that these mutations can play a role in the development of resistance through both loss of target protein expression and interference within the anti-CD20 binding site. These studies help to characterize mechanisms of resistance to mosunetuzumab that could be extended to other anti-CD20 targeted agents. [1] Schuster et al. Journal of Clinical Oncology 2022, 40 (16), 7526.

Classic Hodgkin Lymphomas Display Neuronal-Glial Lineage Transdifferentiation

The molecular drivers of classic Hodgkin lymphoma (HL) underlying its pathologic and clinical distinctions from non-Hodgkin lymphoma (NHL) have been challenging to uncover. The study of HL has been impeded by the rarity of the Hodgkin-Reed-Sternberg (HRS) cells within a dense tumor microenvironment (TME). Previously, we developed a FACS approach to isolate the HRS cells and profiled their whole exomes and genomes (Reichel et al 2015; Maura et al 2023). These studies did not reveal an HRS-specific mutational signature, but rather mutations that overlap with those of NHL, such as those in SOCS1 and HIST1H1E. These data suggested that non-genetic factors such as the underlying cell state may cooperate with somatic mutations to induce HL. However, we previously lacked the ability to co-map somatic mutations and cell states within the same cancer cells. We thus developed Genotyping of Transcriptomes (GoT; Nam et al 2019) that links single-cell RNA-seq (scRNA-seq) and somatic mutations within the same thousands of individual cells. We applied GoT to primary HL samples (n = 10 HL; 68291 cells, Fig. 1A) after FACS-isolation of HRS cells. We analytically integrated the cells across the samples based on gene expression and identified the expected cell types including B-cells, T-cells, monocytes and HRS cells (Fig. 1B). As expected, the HRS cells displayed low expression of B-cell antigens ( CD19, CD79A) and high levels of TNFRSF8 (CD30), CCL17, CCR7 and PD-L1 (Fig. 1C). GoT data revealed that mutations were restricted to the HRS cells (Fig. 1D). Copy number variation (CNV) analysis further confirmed that CNVs were specific to the HRS cells (Fig. 1E). As scRNA-seq has unveiled marked intratumoral cell state heterogeneity across cancers, we posited that the HRS cells may display heterogeneous cell states. Indeed, we identified distinct cell states (Fig. 1F), including cycling HRS and metabolically-active HRS cells upregulating the translation machinery ( NACA, RACK1). We also observed a cell state with elevated inflammatory signals ( TNF, JUN, FOS, CCL22). One group demonstrated a partially preserved B cell program with high CD19 and CD79A. Unexpectedly, we also identified an HRS state with features of neuronal-glial (NG) transdifferentiation, including genes involved in synapse formation ( NRG2, NRXN3). Consistently, genes upregulated in the NG state were enriched in nervous system and neuronal development pathways (Fig. 1G). We further assessed NG gene expressions across normal cell types and confirmed their specificity to NG lineage cells (Fig. 1H). These findings were consistent with dendritic processes of HRS cells highlighted by CD15 and CD30 (Fig. 1I, red arrows). Areas of CD15/CD30 staining devoid of nucleus (Fig. 1I, blue arrows) corroborated the presence of dendritic processes. These results suggested that the NG differentiation may be a key defining feature of HRS cells, whereby dendritic processes facilitate a tight control of the TME. Consistently, we identified strong HRS-immune complexes that persisted through tissue processing and sorting (Fig. 1F). To determine the degree of plasticity or heritability of the HRS cell states, we integrated VDJ sequencing with GoT (GoT-VDJ), whereby somatic hypermutations in the VDJ served as endogenous barcodes. Phylogenetic reconstructions revealed that HRS cell states were distributed across clades (Fig. 1J), unveiling a plastic interchange of HRS cell states. Consistently, GoT showed that mutant cells were present across the cell states (Fig. 1K), indicating that the cell states were decoupled from the genetic identities. We therefore postulated that the NG program may be encoded epigenetically. We performed single nuclei ATAC-seq on HRS-enriched HL samples (n = 5 samples; Fig. 1L). Compared to admixed B cells, the HRS cells displayed enhanced accessibility for binding motifs of the NG lineage transcription factors (TF), e.g. OLIG1/2 and SOX family (Fig. 1M), revealing the TF networks that govern the lineage infidelity program. We further identified that the NG program is cell intrinsic and at least partially stable through in vitro passaging, as 4 of 5 HL cell lines exhibited the highest NG gene expression compared to NHL cell lines (Fig. 1N). Altogether, these single-cell multi-omics studies indicated that an epigenetically encoded NG program may cooperate with lymphoma driving mutations to drive the development of HL, paving novel avenues of therapy.

Redundancy of Glycogen Synthase Kinase 3 in Lymphoma Cell Viability, Proliferation, and the Cytotoxicity of Elraglusib

Introduction Glycogen synthase kinase 3 (GSK3), a ubiquitously expressed serine/threonine kinase, is considered a therapeutic target in non-Hodgkin lymphomas (NHL) due to the poorer survival of patients with higher tumor expression, and the previously observed lethality of GSK3 ‘knock-out’ lymphoma cell lines. Elraglusib (formerly 9-ING-41), a small molecule reported to inhibit the GSK3β paralog, reduces proliferation and induces apoptosis in NHL cell lines, and is undergoing early-phase clinical trials (Wu et al, Blood 2019). However, in recent cell culture studies, the anti-lymphoma effects of elraglusib could not be phenocopied by structurally unrelated, small-molecule GSK3 inhibitors, raising questions about the importance of GSK3-dependent pathways in lymphoma biology and treatment (Coats et al, Cell Commun. Signal 2023). To further validate the lack of impact of GSK3 reduction on lymphoma viability and improve our understanding of the pharmacological properties of elraglusib, we have investigated the effects of reducing GSK3α, GSK3β, or both paralogs concomitantly in lymphoma cells, using paralog-specific and dual GSK3A/B genetic ablation techniques. Additionally, we performed cell-free kinase assays to explore the relative sensitivity of GSK3α and GSK3β as targets of elraglusib. Methods In the lymphoma cell line Karpas-299 (K299), single paralog GSK3A and GSK3B knockdowns (KD) were generated using commercial lentiviral shRNA transfection with subsequent puromycin-selection. Dual paralog KD (both GSK3A and GSK3B)were generated by transfecting stable GSK3A KD cells with a second commercial GSK3B lentiviral shRNA and subsequent neomycin-selection. KD efficiency was quantified by total-GSK3 immunoblot and apoptosis by poly (ADP-ribose) polymerase (PARP) cleavage immunoblot. Cell viability and proliferation were assessed by MTS assay following 72-hour incubations in complete media (RPMI 1640, 10% FBS, 100units/ml penicillin/100µg/ml streptomycin). Paralog-specific cell-free kinase assays were performed using 32P-γ-ATP, phospho-glycogen synthase-peptide-2 substrate and purified recombinant GSK3α and GSK3β. Results Viable and stable K299 single-paralog ( GSK3A or GSK3B) and dual-paralog ( GSK3A and GSK3B) KD cell lines were obtained. KD efficiency was quantified by immunoblot and compared to the parental cell line control for GSK3α (≈30% of control), GSK3β (≈20% of control) and dual GSK3αand GSK3β KD (≈35% control and ≈15% control respectively). All KD cell lines proliferated similarly to parental cell line cells by MTS assay (control vs GSK3B KD normalized to control, mean difference -5.1% (95% CI -11.7 to +1.37 p=0.14), control vs GSK3A/B KD +3.67% (95% CI -2.87 to +10.2 p=0.348)). Elraglusib 5µM induced apoptosis with a significant increase in cleaved/intact PARP (2.35x, p<0.0001) and reduced cell viability in K299 parental cell line and dual GSK3A/B KD cells (IC 50 0.158µM parental control vs 0.124µM dual GSK3A/B KD). The equivalence of paralog inhibition (GSK3β IC 50 0.494µM and GSK3α IC 50 0.446µM) was confirmed in cell-free 32P-γ-ATP kinase assays. Conclusion K299 cells do not depend upon high levels of GSK3 for survival or proliferation. This data extends previous findings regarding the redundancy of GSK3-dependent signalling in lymphoma biology and further suggests that limited therapeutic benefit may be derived from drugs specifically targeting the activity or expression of this protein kinase. The IC 50 of elraglusib remains similar despite a significant reduction in levels of GSK3, again suggesting that GSK3 is not its cytotoxic target. Importantly, elraglusib retains therapeutic potential in lymphoma however GSK3 is not a surrogate marker for its efficacy and additional studies are required to elucidate the mechanisms through which it mediates its cytotoxic effects.

Elraglusib (formerly 9-ING-41) possesses potent anti-lymphoma properties which cannot be attributed to GSK3 inhibition

Elraglusib (formerly 9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3β (GSK3β) undergoing clinical trials for the treatment of various cancers including non-Hodgkin lymphoma (NHL). The drug reduces proliferation of several NHL cell lines and has efficacy in xenograft models of the disease. To confirm the importance of its action on GSK3β, we treated 3 lymphoma cell lines with selective, structurally distinct GSK3 inhibitors: CT99021, SB216763, LY2090314, tideglusib, and elraglusib. Stabilization of β-catenin and reduced phosphorylation of CRMP2, two validated targets of GSK3, were used as functional read-outs for GSK3 inhibition. CT99021, SB216763, and LY2090314 failed to reduce proliferation or viability in any cell line at concentrations that stabilized β-catenin and reduced CRMP2 phosphorylation. There was partial reduction of CRMP2 phosphorylation but no significant effect on β-catenin at cytotoxic doses of elraglusib. There was no indication of GSK3 inhibition at doses of tideglusib that affected cell viability and apoptosis. Cell-free kinase screening confirmed several other targets of elraglusib, distinct from the GSK3 inhibitors with no anti-lymphoma actions, including PIM kinases and MST2. These data question GSK3 as the target of elraglusib in lymphoma, and hence the utility of GSK3 expression as a ‘stand-alone’, therapeutic biomarker in NHL.7CsbmemxsJeEQuU9mL3kzMVideo

LP-284, a small molecule acylfulvene, exerts potent antitumor activity in preclinical non-Hodgkin's lymphoma models and in cells deficient in DNA damage repair

Despite advances in therapies treating non-Hodgkin's lymphoma (NHL), 20~40% of patients experience relapsed or refractory disease. While solid tumors with homologous recombination deficiencies have been successfully targeted with synthetic lethal agents such as poly-ADP ribose polymerase (PARP) inhibitors, such synthetic lethality strategy has not yet been approved to treat patients with NHL. Here we investigated the mechanism of action (MoA) and therapeutic potential of a new-generation acylfulvene compound, LP-284, in both in vitro and in vivo NHL models. One of LP-284's MoA includes inducing the repair of double-strand DNA break (DSB). We found that LP-284 exerts nanomolar potency in a panel of hematological cancer cell lines including fifteen NHL cell lines. In vivo, LP-284 treatment prolongs the survival of mantle cell lymphoma (MCL) cell line JeKo-1 derived xenograft mice by two-fold and shows increased efficacy over bortezomib and ibrutinib. In addition, LP-284 is capable of inhibiting tumor growth of JeKo-1 xenografts that are refractory to bortezomib or ibrutinib. We further showed that LP-284 is particularly lethal in cells with deficient DNA damage response and repair, a targetable vulnerability in NHL.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: We previously performed global miRNA expression profiling of cHL vs. non-Hodgkin lymphoma (NHL) cell lines and sorted germinal centre B-cells (GCB). In the group of 79 deregulated miRNAs (p < 0.05) we became particularly interested in the upregulated miR-23a-3p, a potential oncomiR, predicted to regulate the expression of TNFAIP3 and the downregulated miR-148a-3p, a potential tumor suppressor miRNA, predicted to regulate the expression of IL15. Therefore, we aimed to decipher the role of miR-23a-3p and miR-148a-3p in cHL pathogenesis. Methods: Expression analyses were performed with TaqMan Advanced miRNA Assays versus two miRNA controls. Laser capture microdissection was used for primary HRS collection. 1,000 (miRNA expression) and 2 × 200 (DNA methylation) HRS cells were collected per case. Bisulfite pyrosequencing assay was designed using the PyroMark Software and sequencing performed using the PyroMark Q24 sequencer. MiRNA-mRNAs interactions were validated with Dual-Glo Luciferase Assay using respective miRNA mimics. For overexpression pre-miRNAs were cloned into the pCDH-CMV-MCS-EF1α-GreenPuro vector and transductions were performed in triplicate. Cell proliferation was measured using CCK-8 in four replications in three independent reactions by the GloMax®96 Microplate reader. Results: We confirmed the overexpression of miR-23a-3p (p < 0.001) and downregulation of miR-148a-3p (p < 0.014) in microdissected HRS cells (n = 10) vs. GCB cells (n = 10). Moreover, we observed inverse correlation of miR-148a-3p expression with DNA methylation level of an adjacent CpG island (r = −0.72, p < 0.01; cHL n = 7, NHL n = 10). Elevated DNA methylation of this CpG island was detected also in 2/6 cHL primary cases. Next, we validated interactions between miR-23a-3p and miR-148a-3p and their target genes. A reduction by 34% (p < 0.001) and 40% (p < 0.01) of the luciferase signal was observed in consequence of the interaction of miR-23a-3p mimic—TNFAIP3 3’UTR and miR-148a-3p mimic—IL15 3’UTR, respectively. Moreover, the abundance of TNFAIP3 protein was reduced after overexpression of the miR-23a (coexpressed with miR-27a) in L428 (p = 0.0018) and GCB6-16 (p = 0.0083) cell lines, compared to empty vector transductions. Whereas overexpression of miR-148a in three cHL cell lines caused a 32% decrease (p < 0.05) in proliferation in the KM-H2 cell line compared to empty vector transductions. The research was funded by: This research was funded by the National Science Center, Poland grants: 2014/14/M/NZ2/00529 to M.G. and the 2019/32/T/NZ5/00441 Etiuda scholarship to JP. SH is supported by the German Research Foundation (DFG, HA6145/3-1). MG, ADz-K and NR received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952304. Keywords: Hodgkin lymphoma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

Characterization of extracellular vesicles in AIDS-associated non-Hodgkin lymphoma.

Abstract Even with the use of anti-retroviral therapy, HIV +subjects develop non-Hodgkin lymphoma (NHL) at a higher frequency than uninfected subjects. Extracellular vesicles (EVs) secreted by cells may contain immunomodulatory molecules; for example, tumor cells can secrete EVs containing bioactive PD-L1 that can inhibit anti-tumor responses. We have shown EVs bearing PD-L1, TNF-RII or IL-6Rα can be biomarkers of NHL risk. Here we aim to characterize EVs isolated from EBV +and EBV −lymphoma cell lines to define their involvement in lymphomagenesis and identify more EV-associated biomarkers for NHL risk (like immune checkpoint molecules CD80, CD86, CD28). EVs were isolated from AIDS-NHL and NHL cell lines using a total exosome isolation kit and analyzed by Western blot. A subset of these were also analyzed by proteomics using Metascape, Reactome, and R. EVs were also isolated from archived serum of HIV +individuals before NHL onset (n = 51) and matched HIV +controls (n = 53) from participants of the MACS/WIHS Combined Cohort study, with a total exosome isolation kit and characterized by Western blot. EV-specific markers were detected in EVs isolated from cell lines and serum. EVs isolated from EBV +cell lines largely expressed LMP1, an EBV CD40 homolog that induces EBV-driven B cell transformation. In EVs isolated from serum, levels of CD80, CD86, or CD28 were comparable between pre-NHL samples and matched controls, though control samples tended to have greater CD80 and CD86 expression. Our findings shed light on the EV marker expression profiles of AIDS-NHL and NHL cell lines and show LMP1 expression in EBV +cell lines. Additionally, this research provides initial insight into the complex composition of EVs and their role in lymphomagenesis. Supported by grants from NIH (R01CA228157, P30CA016042-S, R01CA228157S)

Abstract CT136: An open-label phase I/IIa study to evaluate the safety and efficacy of CCS1477 as monotherapy and in combination in patients with advanced hematological malignancies

Abstract Background Inobrodib (CCS1477) is a first in class potent, selective, and orally bioavailable inhibitor of the bromodomains of p300 and CBP, two closely related histone acetyl transferases with oncogenic roles in hematological malignancies. In pre-clinical studies inobrodib potently inhibited cell proliferation in acute myeloid leukemia (AML), multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) cell lines. Inobrodib demonstrated dose-dependent efficacy in corresponding xenograft models, eliciting tumor regression at the highest doses. These effects were accompanied by significant reductions in expression of MYC, MYB and IRF4. Low doses of inobrodib also demonstrated good efficacy when added to standard-of-care treatments, providing a strong rationale for combination treatment in patients. Inobrodib represents a novel and differentiated approach and offers a potential new therapeutic option for patients who have relapsed or are refractory to current standard of care therapies in AML, higher risk MDS, MM or NHL. Study Design and Methods NCT04068597 is an adaptive multi-arm/multi-stage trial allowing exploration of p300/CBP inhibition as monotherapy or in combination with different agents across multiple indications including AML/higher risk MDS, MM, and NHL. The trial was initiated as a dose escalation rolling 6 design separately in AML/MDS and MM/NHL to ensure tolerability is explored separately across groups of indications, with the possibility to expand in monotherapy across different indications. Key inclusion criteria include patients with confirmed relapsed or refractory disease, with patients having received standard therapy (typically at least two prior lines of therapy). Response criteria are assessed using standard methodologies for the different indications. Blood and tumor samples are collected for exploratory biomarker analysis to understand mechanisms of response to treatment or disease progression. The recommended phase 2 dose/schedule has been determined, and monotherapy expansion arms are currently open in selected indications. Cohorts investigating dose escalation with standard of care agents pomalidomide/dexamethasone in MM as well azacytidine with or without venetoclax in AML or MDS are currently enrolling, with the possibility of expansion for promising combinations. An additional ‘rescue’ cohort for MM patient who failed inobrodib monotherapy will be used to determine whether addition of pomalidomide with or without dexamethasone may reverse resistance to inobrodib. This cohort will also explore tolerability and clinical activity of steroid-sparing combination regimen. Citation Format: Tomasz Knurowski, Emma Searle, Karen Clegg, Neil Pegg, William West, Debbie Haynes, Kristopher Frese, Tim C. Somervaille. An open-label phase I/IIa study to evaluate the safety and efficacy of CCS1477 as monotherapy and in combination in patients with advanced hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT136.

Identification of pyrrolo[3′,4’:3,4]cyclohepta[1,2-d][1,2]oxazoles as promising new candidates for the treatment of lymphomas

Unsatisfactory outcomes for relapsed/refractory lymphoma patients prompt continuing efforts to develop new therapeutic strategies. Our previous studies on pyrrole-based anti-lymphoma agents led us to synthesize a new series of twenty-six pyrrolo[3′,4':3,4]cyclohepta[1,2-d] [1,2]oxazole derivatives and study their antiproliferative effects against a panel of four non-Hodgkin lymphoma cell lines. Several candidates showed significant anti-proliferative effects, with IC50's reaching the sub-micromolar range in at least one cell line, with compound 3z demonstrating sub-micromolar growth inhibitory effects towards the entire panel. The VL51 cell line was the most sensitive, with an IC50 value of 0.10 μM for 3z.Our earlier studies had shown that tubulin was a prominent target of many of our oxazole derivatives. We therefore examined their effects on tubulin assembly and colchicine binding. While 3u and 3z did not appear to target tubulin, good activity was observed with 3d and 3p.Molecular docking and molecular dynamics simulations allowed us to rationalize the binding mode of the synthesized compounds toward tubulin. All ligands exhibited a better affinity for the colchicine site, confirming their specificity for this binding pocket. In particular, a better affinity and free energy of binding was observed for 3d and 3p. This result was confirmed by experimental data, indicating that, although both 3d and 3p significantly affected tubulin assembly, only 3d showed activity comparable to that of combretastatin A-4, while 3p was about 4-fold less active.Cell cycle analysis showed that compounds 3u and especially 3z induced a block in G2/M, a strong decrease in S phase even at low compound concentrations and apoptosis through the mitochondrial pathway. Thus, the mechanism of action of 3u and 3z remains to be elucidated.Very high selectivity toward cancer cells and low toxicity in human peripheral blood lymphocytes were observed, highlighting the good potential of these agents in cancer therapy and encouraging further exploration of this compound class to obtain new small molecules as effective lymphoma treatments.

Identification of two unannotated miRNAs in classic Hodgkin lymphoma cell lines.

MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1.

Co-Transduced CD19/BCMA Dual-Targeting CAR-T Cells for the Treatment of Non-Hodgkin Lymphoma

CAR-T cells targeting CD19 have accomplished impressive responses in relapsed/refractory non-hodgkin lymphoma (NHL) patients, but disease recurrence is a concerning hurdle to overcome. This complication is mainly due to a selective pressure on tumor cells expressing CD19, which leads to loss of expression or downregulation to levels that cannot be detected by CAR-T cells. Simultaneous targeting of more than one antigen has been proposed as a compelling strategy to prevent tumor escape. CD19 is expressed along B-cell differentiation, and BCMA is expressed not only on plasmatic cells but also on mature B-cells. Thus, most NHL subtypes express both CD19 and BCMA. Here, we hypothesized that dual CD19 and BCMA targeting could reduce tumor antigen escape and enhance the antitumor efficacy of CAR-T cells. CAR constructs targeting CD19 (ARI0001) or BCMA (ARI0002h) were engineered in our institution and both have demonstrated safety and efficacy for treating patients with B-cell lymphoid neoplasms and multiple myeloma, respectively. Both CARs contain the 41BB co-stimulatory domain in tandem with the CD3z. We tested the antitumoral efficacy of three different dual-targeting CD19/BCMA strategies: 1) a bicistronic vector encoding both CAR constructs separated by a T2A cleavage peptide (ARI0001-T2A-ARI0002h); 2) co-transduced T cells with ARI0001 and ARI0002h lentiviral vectors to obtain a product with three CAR populations comprising both single-CARs and a double positive population; 3) pooled ARI0001 and ARI0002. Conventional functional assays such as cytokine secretion and specific lysis after short-term co-culture of CAR-T cells with NHL cell lines co-expressing CD19 and BCMA at constitutive levels revealed that all strategies were active but, did not discriminate between single- and dual-CARs groups. In order to mimic a potential relapse due to CD19 downregulation, we developed a CD19low density cell line of Burkitt lymphoma through its expression under a weak promoter. After 7-days of co-culture of CAR-T cells with RAMOS-CD19Low, only the co-transduction product maintained tumor elimination. Next, we analyzed the capacity of single versus dual CAR-T cells to kill malignant B cells and release cytokines in co-culture with NHL Patient Derived Lymphoma Spheroids. We tested samples from patients with 1) follicular lymphoma, 2) diffuse large B cell lymphoma (DLBCL), and 3) DLBCL patients who relapsed or had refractory disease post-CD19 CAR-T cell treatment. Under these more challenging conditions, differences among effector groups were maximized. ARI0001 and bicistronic CAR-T cells displayed similar effector functions in terms of killing capacity and cytokine production, especially when CD19 was expressed at high densities. ARI0002h presented antitumor efficacy but not as high as ARI0001. Co-transduction and pool products showed increased cytokine production and killing, being capable of B-cell elimination in all cases. A high disease burden of Burkitt lymphoma in vivo model revealed that co-transduction product significantly increased mice survival compared to ARI0001. This increase in survival was even more remarkable when the model was CD19low. No significant differences were observed between the co-transduction and pool groups. In summary, we propose a relatively simple preparation of a dual-CAR-T cells targeting CD19 and BCMA, which is particularly effective against NHL cell lines expressing low CD19 densities. Co-transduction is advantageous with respect to the pool since it only requires one manufacturing process, decreasing the cost of the therapy and increasing patients' access to these novel therapies. Our studies provide a strong rational to test CD19/BCMA dual-CAR-T cells in clinical trials for the treatment of NHL patients.

A CD79b Targeting ADC with Superior Anti-Tumor Activity and Safety Resulting in Significantly Improved Therapeutic Index (TI): Safe and Efficacious CD79b ADC

The Araris' site-specific and 1-step enzymatic linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC (ARC-02) with this technology showing a higher therapeutic index (TI) compared to the approved ADC polatuzumab-vedotin (Polivy®) in different preclinical models. Therefore, ARC-02 is considered a promising next generation treatment alternative for patients suffering from Non-Hodgkin lymphoma (NHL). Using native polatuzumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24 hours the highly homogeneous and pure ARC-02 development candidate with a well-defined drug-to-antibody ratio (DAR) of 1.9 and with high purity (monomer content > 98%). ARC-02 is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In-vitro ARC-02 demonstrated potent cytotoxicity in four different NHL cell lines tested so far with similar potency (EC50 values) compared to the approved Polivy® ADC despite of the lower toxin load of ARC-02 (DAR of 1.9) compared to Polivy® (DAR of 3.5). ARC-02 clearly differentiates from Polivy® by extraordinary stability in mouse, cynomolgus monkey and human sera exemplified by the absence of payload deconjugation or linker cleavage under physiological conditions (at 37°C) and long-term incubation (>2 weeks). Exceptional stability could also be confirmed in vivo in pharmacokinetic studies in mice, rats and cynomolgus monkeys demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab parental antibody without premature toxin deconjugation in the circulation resulting in improved pharmacokinetics of ARC-02 compared to Polivy® (half-life in mice 10 days for ARC-02 vs 5 days for Polivy®). Despite the excellent stability of ARC-02 in vitro and in vivo, the MMAE toxin is still efficiently released from ARC-02 in lysosomal human Cathepsin B cleavage or human liver-lysosome release assays which is providing the foundation for the observed robust anti-tumor activity in vivo: ARC-02 was compared head-to-head to Polivy® in two different CD79b-expressing established tumor models in vivo (Granta-519 and Ramos). Single dose treatment of ARC-02 at very low payload doses induced effective and long-lasting anti-tumor responses with complete tumor eradication achieved at about half of the Polivy® payload dose in both NHL established tumor models. Based on these in vivo efficacy experiments, the minimal effective dose (MED) of ARC-02 that is still inducing eradication of established tumors is determined to be as low as 10 μg/kg based on the MMAE payload dose or 1 mg/kg based on the total ADC dose. Finally, the highest non-severely toxic dose (HNSTD) of ARC-02 in repeat dose toxicology studies was determined to be ≥30 mg/kg and ≥24 mg/kg in rats and cynomolgus monkeys, respectively, which is substantially higher than the HNSTD reported for Polivy® (i.e. 10 mg/kg in the rat and 3 mg/kg in the cynomolgus monkey as described in the Polivy® Biologic License Application). Therefore, the tolerability in rodents and non-human primates is at least 3 and 8 times higher when compared to historical safety data of Polivy®. Using the HNSTD derived from repeat dose non-human primate toxicity study (24 mg/kg) and the MED calculated from single dose rodent efficacy studies (1 mg/kg) an impressive TI of 24 can be calculated for ARC-02 which is substantially higher than the TI of Polivy®. These encouraging results obtained so far indicate that ARC-02 a) has very favorable biophysical properties, b) shows a consistent and highly defined DAR, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) has improved tolerability in rodents and non-human primates and f) shows an impressive improvement in TI and ultimately warrant further development of ARC-02.

B-Cell Targeting Anti-CD37 Humanized Antibodies Engineered for Potent Effector Functions and Extended Plasma Half-Life

Background: CD37 is a tetraspanin protein abundant on the surface of B-cells but absent in hematopoietic stem cells and plasma cells. Its expression pattern has attracted interest as a therapeutic target for B-cell malignancies including non-Hodgkin lymphomas (NHL), a heterogeneous disease with substantial unmet clinical need due to relapse and resistance to currently available treatments. During the last years, different approaches have been followed to exploit CD37 in targeting malignant B-cells, such as the generation of CAR-T cells and monoclonal antibodies. Of note, the anti-CD37 antibody-drug conjugates (ADC) naratuximab emtansine and the CD37-targeting radioimmunotherapy (RIT) Betalutin have shown efficacy in NHL patients during clinical testing. Here we report on the design of humanized anti-CD37 IgG1 antibodies (NNV029, NNV031 and NNV032) that are equipped with a unique Fc technology and N-glycan engineering for enhanced effector functions and extended plasma half-life. Methods: The humanized anti-CD37 candidates were characterized in vitro and in vivo and benchmarked against the anti-CD20 antibodies approved for NHL treatment, rituximab and obinutuzumab, and a recombinant version of the bi-paratopic anti-CD37 antibody DuoHexaBody-CD37, currently in Phase I clinical development. We assessed their Fcγ receptor binding properties and their ability to induce antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) against CD37-expressing NHL cell lines and patient-derived chronic lymphocytic leukemia (CLL) cells. Moreover, we evaluated their human FcRn binding and transport properties using the human endothelial recycling assay (HERA) followed by studies in human FcRn (Tg32) expressing mice. Lastly, we compared the therapeutic efficacy of the humanized antibodies versus obinutuzumab and recombinant DuoHexaBody-CD37 in a xenograft disseminated Daudi tumor model in Tg32 mice. Results: We revealed that the designed anti-CD37 IgG1 candidates have higher affinity towards FcγRIIa expressed on macrophages and that afucosylation enhanced FcγRIII engagement. Accordingly, using cell-based effector assays, potent ADCP and ADCC were measured. In NHL Burkitt's lymphoma cell lines, ADCP induction by the anti-CD37 humanized antibodies was higher than that displayed by obinutuzumab, rituximab and recombinant DuoHexaBody-CD37, while in DLBCL and MCL cell models it was in a similar range or slightly lower than that shown by the benchmarking antibodies. Moreover, the afucosylated version promoted very strong ADCC, which was superior to that of recombinant DuoHexaBody-CD37 and rituximab, but comparable to that of the afucosylated antibody obinutuzumab. In contrast, all the humanized anti-CD37 IgG1 candidates displayed very modest CDC activation. Fc-engineering for enhanced human FcRn binding was shown to increase human FcRn-mediated rescue from intracellular degradation, which translated into prolonged plasma half-life in Tg32 mice of the anti-CD37 IgG1 antibodies. Finally, all the designed anti-CD37 antibodies performed significantly better than vehicle control treatment in treating Daudi-induced disease in vivo, showing equal therapeutic effect as that of obinutuzumab and recombinant DuoHexaBody-CD37. Conclusions: Taken together, the humanized and Fc-engineered anti-CD37 antibodies show potent effector functions, favorable pharmacokinetic properties and in vivo therapeutic efficacy. As such, the designed CD37 antibodies should be attractive modalities in targeted treatment of B cell malignancies, such as NHL, including lymphomas non-responding to anti-CD20 treatments, but also potentially for B-cell driven autoimmune disorders.

Dampening of cytotoxic innate lymphoid cells: A new tumour immune escape mechanism in B cell non-Hodgkin's lymphoma

The role and regulation of innate immune cells is poorly understood in B-cell non-Hodgkin lymphoma (NHL). As natural killer (NK) cells, helper innate lymphoid cells (ILCs) are lymphocytes endowed with either anti- or pro-tumour activity and involved in inflammatory processes.In our ex vivo analysis of NK cells and ILCs from NHL patients, we observed that, in comparison to healthy donors (HD), the frequency of the cytotoxic subset of NK cells, the CD16+ NK, decreased in patients’ peripheral blood. In general, circulating NK cells showed a pro-tumorigenic phenotype, while ILCs displayed a more activated/cytotoxic phenotype. Conversely, at the tumour site, in patients’ lymph nodes, ILCs showed a low expression of granzyme.In vitromixed lymphocyte-tumour cell cultures with HD PBMCs and NHL cell lines demonstrated that ILC cytotoxic potential was lowered by the presence of tumour cells but, in the absence of T regulatory cells (Tregs), their cytolytic potential was recovered.Our data shed novel light on dysfunctional innate immunity in NHL. We suggest a new mechanism of tumour immuno-escape based on the reduction of cell cytotoxicity involving ILCs and likely controlled by Tregs.

Abstract A06: Strategies to circumvent resistance to cyclin-dependent kinase-9 inhibition (CDK9i) in non-Hodgkin lymphoma (NHL)

Abstract Introduction Oncogenic programs are facilitated by activators of transcriptional machinery, including certain CDKs. CDK9, a component of the positive transcription elongation factor b (pTEFb) complex, has arisen as an attractive target due to its regulation of MYC and MCL1 transcription (Hashiguchi et al, 2019). Nevertheless, we and others have observed resistance to CDK9i in vitro and in vivo. Here we studied the effects of CDK9 inhibition using the novel selective CDK9 inhibitor AZD4573, currently under evaluation in clinical trials. Methods A panel of NHL cell lines (OCI-LY3/19, SUDHL4/10/16, VAL, U2932) and primary NHL cells were employed. Response to CDK9i was characterized using LC-MS proteomic analysis, RNA-Seq, and CRISPR-Cas9 Screening. Results NHL cells treated with AZD4573 for 6h exhibited a dose dependent reduction in phospho-RNAPIISer2, as well as loss of MYC and Mcl-1. CDK9i potently inhibited proliferation and induced apoptosis in a panel of NHL cell lines (IC50 range 5-30 nM). Two DLBCL cell lines underwent LC-MS proteomic analysis following AZD4573 treatment (30 nM, 3h). Treated cells exhibited rapid loss of MYC, Mcl-1, PIM3 and JUNB protein levels. We observed broad transcriptional repression via RNA-seq, including downregulation of PIM3 and JUNB (30 nM, 3h). However, a subset of genes, including MYC, PIM1 and JUNB underwent early transcriptional recovery, confirmed by immunoblotting, thus identifying candidate genes which may account for resistance to CDK9i. PIM kinases cooperate with the PI3K/ATK signaling pathway, and have been proposed as therapeutic targets in cancer. We next used SGI1776 (PIM1 specific) and AZD1208 (pan-PIM) in combination with AZD4573, and found synergy between them in a panel of 4 cell lines and primary samples. OCI-LY3 xenograft mice treated with a combination of AZD4573 (15 mg/kg; IP; once weekly) and AZD1208 (30 mg/kg; oral gavage, twice weekly) demonstrated restricted tumor growth and increased survival compared to control. To further understand pathways mediating resistance to CDK9i, we carried out a genome-wide loss of function CRISPR-Cas9 library screen. Two Cas9-expressing NHL cell lines were transduced with a CRISPR library comprised of ~5 unique sgRNA per gene. Loss of AKT, RPTOR, or mTOR, among others, sensitized cells to AZD4573. Concurrent treatment with PI3K inhibitors synergistically suppressed proliferation of NHL cell lines and primary cells treated with AZD4573 in vitro. OCI-LY3 xenograft mice were treated with AZD4573 (15 mg/kg; IP; once weekly), Copanlisib (15 mg/kg; IP; twice weekly), or a combination of both. Combo treatment restricted tumor growth and prolonged survival to a greater extent than either drug alone. Conclusions CDK9i with AZD4573 downregulated numerous oncoproteins. However, a subset of genes including MYC and PIM3 recovered transcription. PI3K/AKT pathway was implicated in resistance to CDK9i in CRISPR library screens. Concurrent targeting of pro-survival pathways (e.g., PIM, PI3K) partially reversed resistance to CDK9i. Citation Format: Elana Thieme, Duanchen Sun, Nur Bruss, Geeta Sharma, Tingting Liu, Daniel Coleman, Tamilla Nechiporuk, Daniel Bottomly, Shannon McWeeney, Patrick Pirrotte, Zheng Xia, Alexey Danilov. Strategies to circumvent resistance to cyclin-dependent kinase-9 inhibition (CDK9i) in non-Hodgkin lymphoma (NHL) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A06.