Lymphoma Pre-clinical Models Research Articles

Constitutive PRMT5 Expression Drives Spontaneous Lymphoblastic Lymphoma In Vivo

Protein arginine methyltransferase 5 (PRMT5) is a highly conserved enzyme that catalyzes monomethylation and symmetric dimethylation of arginine residues on many target proteins, including histones and non-histone proteins (P53, P65/NFκB, E2F1, AKT and spliceosome proteins). (Shailesh, 2018) PRMT5 is overexpressed in numerous solid and hematologic malignancies. (Yang, 2013) It is associated with several oncogenic mechanisms, from driving the malignant phenotype to promoting tumor cell survival and invasiveness. While preliminary data has shown PRMT5 to be upregulated in T-ALL/LBL, the mechanism by which PRMT5 promotes T-ALL/LBL has yet to be established. (Mei, 2019) Preclinical transgenic models of leukemia and lymphoma driven by a variety of oncogenes (MYC, NOTCH) have identified PRMT5 activity as a critical component for the driver phenotype. (Li, 2015) To test the hypothesis that overexpression of this enzyme is capable of malignant driver activity, we developed a transgenic (Tg) mouse model designed to over-express PRMT5 in the lymphoid compartment. (Figure 1) Tg mice were created by injecting a linearized pBH-Eμ-PRMT5 vector into FVB/N pronuclei and implanted into pseudo-pregnant FVB/N mice. We obtained five founder lines crossed with wild-type FVB/N mice to obtain the F1 generation. RT-PCR for PRMT5 showed preferential overexpression in Tg B lymphocytes compared to control FVB/N mice (p<0.01) but also overexpression in T and NK cell compartments due to leaky Eμ phenotype. We observed a high percentage of F1 progeny from all five founder lines to develop spontaneous lymphoproliferative disease compared to control mice (p<0.001). Immunophenotypic and pathologic evaluation was performed by flow cytometry, immunohistochemistry, and clonality by IgH and TCR Vβ PCR analysis. Pathologic features showed a range of lymphoid neoplasms, with pre-T lymphoblastic immunophenotype being the most common. Tumor-bearing animals demonstrated diffuse involvement of all organs, peripheral blood, and bone marrow. Pre-T lymphoblastic lymphomas (LBL) showed expression of cytoplasmic CD3, surface CD4 (dim), TdT, and variable Ki67 with constitutive overexpression of PRMT5, MYC, CYCLIN D1, Ki67, and CD99. We developed a T-LBL cell line (Tg813) derived from a thymic tumor that displayed clonal TCR Vβ.17 gene rearrangement, intracellular TdT and CD3 expression with dim surface CD4 expression, immunophenotypic characteristics consistent with T-ALL/LBL. This cell line was maintained in culture and successfully passaged in immune-competent FVB mice, which led to disseminated disease, suggesting that PRMT5 drives an immune escape program in vivo. Preliminary data demonstrate that tumor-bearing animals display a prominent population of normal and neoplastic T-cell subsets with strong PD1 expression. PD1 expression in T-ALL/LBL has been linked with oncogenic function (Yang, 2019) and, in normal T-cell subsets, is a marker for exhaustion. (Lee, 2015) In addition, treatment of our Tg813 in vitro model with PRMT5 knockdown (CRISPR CAS9) and selective inhibition with a SAM-competitive inhibitor (C220 and PRT382, Prelude Therapeutics) showed a reduction of global symmetric dimethyl arginine, loss of the PRMT5 epigenetic mark H4R3(Sme2) and decreased proliferation with cell cycle arrest by day 6, findings consistent with PRMT5-dependent driver activity. Engraftment of Tg813 into SCID and immunocompetent FVB/N mice led to disseminated lymphomas (mean: 21 days). Tg813 engrafted animals treated with C220 (10mg/kg) showed improved survival compared to vehicle-treated mice (p<0.001). This preclinical model supports the hypothesis that PRMT5 over-expression provides cancer driver activity and a unique opportunity to study the role of this oncogene in an immune-competent, in vivo model system. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract A02: Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981)

Abstract SUMOylation is a post-translational modification that leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. TAK-981, a novel selective inhibitor of SUMO-activating enzyme (SAEi) has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of non-Hodgkin lymphoma (NHL). TAK-981 was provided by Takeda (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL lines and led to rapid protein de-SUMOylation in a dose-dependent manner. TAK-981 induced pronounced DNA break in DLBCL cells (comet assay) and cell cycle arrest (propidium iodide). RNA-Seq and ATAC-Seq analysis of TAK-981-treated OCI-LY3 and U-2932 cells revealed upregulation of the NFκB, and OXPHOS signaling pathways. We noted that mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (EM) and a change of mitochondrial dynamics (ICC for p-Drp1), accompanied by rapid accumulation of reactive oxygen species. Seahorse respirometry revealed dramatic downmodulation of OXPHOS. Metabolomic profiling of DLBCL cells treated with TAK981 exhibited downmodulation of TCA substrates. Exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53 and DNA damage pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981 exposed splenocytes exhibited a reduction of maximal respiration by Seahorse. In summary, pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction and metabolic reprogramming. Citation Format: Tingting Liu, Vi Lam, Duanchen Sun, Elana Thieme, Tamilla Nechiporuk, Daniel Bottomly, Olga V Danilova, Nur Bruss, Martina Cusan, Lili Wang, Shannon K McWeeney, Jeffrey W Tyner, Steve Kurtz, Zheng Xia, Alexey Danilov. Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981) [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 A02.

Off-the-shelf third-party HSC-engineered iNKT cells for ameliorating GvHD while preserving GvL effect in the treatment of blood cancers

SummaryAllo-HSCT is a curative therapy for hematologic malignancies owing to GvL effect mediated by alloreactive T cells; however, the same T cells also mediate GvHD, a severe side effect limiting the widespread application of allo-HSCT in clinics. Invariant natural killer T (iNKT) cells can ameliorate GvHD while preserving GvL effect, but the clinical application of these cells is restricted by their scarcity. Here, we report the successful generation of third-party HSC-engineered human iNKT (3rdHSC-iNKT) cells using a method combining HSC gene engineering and in vitro HSC differentiation. The 3rdHSC-iNKT cells closely resembled the CD4−CD8−/+ subsets of endogenous human iNKT cells in phenotype and functionality. These cells displayed potent anti-GvHD functions by eliminating antigen-presenting myeloid cells in vitro and in xenograft models without negatively impacting tumor eradication by allogeneic T cells in preclinical models of lymphoma and leukemia, supporting 3rdHSC-iNKT cells as a promising off-the-shelf cell therapy candidate for GvHD prophylaxis.

S216: CLINICAL ACTIVITY OF CC-99282, A CEREBLON E3 LIGASE MODULATOR (CELMOD) AGENT, IN PATIENTS (PTS) WITH RELAPSED/REFRACTORY NON-HODGKIN LYMPHOMA (R/R NHL) – RESULTS FROM A PHASE 1, OPEN-LABEL STUDY

Background: CC-99282 is a novel, oral small molecule CELMoD® agent that co-opts cereblon to induce targeted degradation of Ikaros/Aiolos, transcription factors critical for development of B-cell malignancies. Compared with immunomodulatory drugs (IMiD®) and other CELMoD agents, CC-99282 had similar immunostimulatory effects and 10- to 100-fold stronger antiproliferative and apoptotic activity in preclinical models of diffuse large B-cell lymphoma (DLBCL). Aims: To evaluate the safety, tolerability, maximum tolerated dose, pharmacokinetics (PK), and preliminary efficacy of CC-99282 in pts with R/R NHL. Methods: CC-99282-NHL-001 (NCT03930953) is a 2-part multicenter first-in-human study comprising dose escalation of CC-99282 monotherapy (part A) and expansion with or without combination partners (part B). Part A includes pts with R/R DLBCL or follicular lymphoma (FL) who had progressed after ≥2 lines of therapy, including IMiD/CELMoD agents and chimeric antigen receptor T-cell (CAR-T) therapy, and pts with R/R DLBCL who received ≥1 line of standard therapy and are unfit for transplant. Pts receive oral CC-99282 0.2, 0.4, 0.6, or 0.8 mg once daily on 3 intermittent dosing schedules of 28-day cycles, with ≥3 pts per dosing cohort. Results: As of Oct 6, 2021, 50 pts were treated in part A (38 DLBCL, 12 FL; median age 66y; 58% male); 17 pts (34%) were ongoing and 26 pts (52%) discontinued due to progressive disease. Grade 3/4 CC-99282–related adverse events were reported in 32 pts (64%); the most common were neutropenia (29 pts [58%]), thrombocytopenia (4 pts [8%]), and anemia (4 pts [8%]). Correlation analysis showed that grade 4 neutropenia in the first month was more strongly associated with number of prior lines of alkylating agent and anti-CD20 therapy than with CC-99282 dose level or schedule. Neutropenia was manageable with CC-99282 dose modifications and granulocyte colony–stimulating factors. All dose-limiting toxicities were hematologic. The maximal recommended doses for schedules of interest were 0.6 mg on 7/14 days and 0.4 mg on 14/28 days. For doses ≥0.4 mg on schedules of interest, the overall response rate was 42% (15/36 evaluable pts; 6 FL, 9 DLBCL), with complete responses in 6 pts and partial responses in 9 pts. Responders included pts previously treated with CAR-T therapy and/or IMiD/CELMoD agents. Responses were durable, with median durations of 239 days (range 48–587; median follow-up [mFUP] 247 days [range 21–690]) and 112 days (range 63–414; mFUP 121 days [range 22–464]) for 7/14- and 14/28-day schedules, respectively. CC-99282 was absorbed rapidly and had a prolonged terminal half-life (median ~50 hours at doses ≥0.4 mg), with considerable distribution into the peripheral compartment. Increase in plasma CC-99282 and degradation of Ikaros/Aiolos in peripheral T cells occurred in a dose-dependent manner, and maximum degradation (>90%) occurred by day 4 of treatment at doses ≥0.4 mg. Within 2 weeks of initiating CC-99282, peripheral T-cell subsets showed a significant shift toward a more activated phenotype (P<0.05). Analysis of mutant circulating tumor DNA levels showed rapid reductions that correlated with response to CC-99282, suggesting strong tumor cell–intrinsic activity. Summary/Conclusion: CC-99282 monotherapy showed a manageable safety profile and demonstrated promising efficacy in heavily pretreated pts with R/R NHL. PK and pharmacodynamic data were consistent with robust and rapid CC-99282 antitumor activity. This study is ongoing, with patients actively being enrolled in the monotherapy and CC-99282+rituximab combination expansion cohorts.

Abstract 1050: Mechanistic studies investigating the synergistic combination of Loncastuximab Tesirine and Ibrutinib in pre-clinical models of B-cell non-Hodgkin lymphoma

Abstract Loncastuximab tesirine-lpyl (formerly ADCT-402) is a pyrrolobenzodiazepine dimer-based antibody-drug conjugate directed against human CD19 which has been recently approved by the United States Food and Drug Administration for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL). Pre-clinically, loncastuximab tesirine has shown potent and specific anti-tumor activity in lymphoma models both as single agent and in combination with other approved drugs. Clinically, loncastuximab tesirine is being tested in multiple clinical trials, either as monotherapy or in combination with other anti-lymphoma drugs including ibrutinib. Ibrutinib is a small molecule inhibitor of Bruton’s tyrosine kinase, approved for treatment of mantle cell lymphoma (MCL), chronic lymphocytic leukaemia and other haematological malignancies.Here, we investigated the combined effect of loncastuximab tesirine (lonca) and ibrutinib in pre-clinical models of B-cell non-Hodgkin lymphoma (NHL).In vitro, the combination of lonca and ibrutinib was tested in a panel of 5 NHL-derived cell lines covering activated B-cell-DLBCL (TMD8 and OCI-Ly3), germinal center B-cell-DLBCL (WSU-DLCL2) and MCL (REC-1 and Granta-519) subtypes and showed synergistic activity in all but one cell line (WSU-DLCL2), as assessed by the Chou-Talalay method.Quantification of cell viability (propidium iodide [PI]-negative and Annexin V-negative), early apoptosis (Annexin V-positive and PI-negative) and late apoptosis (Annexin V-positive and PI-positive) showed a statistically significant reduction of viable cells accompanied by an increase in early apoptotic cells after 72-hour treatment with the combination compared to the single agents.Analysis of γH2AX and cleaved PARP protein expression revealed that ibrutinib drives the increase in both γH2AX and cleaved PARP after 24 hours of treatment, while lonca is largely responsible for the upregulation of both markers after 72 hours of treatment, suggesting that the two drugs work with different kinetics. Interestingly, increased γH2AX was detected in the combination setting compared to the two single agents at the 72-hour time-point.Gene expression profiling of TMD8 cells treated with lonca, ibrutinib or their combination revealed that the cytokine-cytokine receptor interaction pathway and the nuclear factor kappa light chain enhancer of activated B cells pathway were among the most affected by the drug combination. Interleukin-10 (IL-10) was one of the most downregulated genes. Interestingly, quantitative gene and protein expression analysis revealed that IL-10 downregulation was more effective in the combination setting compared to the single agents. In conclusion, these preclinical data support the ongoing clinical investigation of lonca and ibrutinib in patients with advanced DLBCL or MCL. Citation Format: Danilo Cucchi, Nikoleta Sachini, Patrick H. van Berkel, Francesca Zammarchi. Mechanistic studies investigating the synergistic combination of Loncastuximab Tesirine and Ibrutinib in pre-clinical models of B-cell non-Hodgkin lymphoma [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 1050.

Abstract 5454: Amdizalisib (HMPL-689), a highly selective PI3Kδ inhibitor, exhibits potent anti-tumor activity in pre-clinical B-cell lymphoma models

Abstract Background: The delta isoform of PI3K (PI3Kδ) plays an important role in B-cell development and function by mediating the signaling of key receptors on B cells. Aberrant activation of PI3Kδ signaling pathway promotes the survival and proliferation of malignant B cells, making selective inhibition of this isoform a promising therapeutic approach for the treatment of B cell malignances. Amdizalisib (HMPL-689, Amdiz), discovered and being currently developed in a pivotal phase II clinical trial (NCT04849351) by HUTCHMED, is a highly potent and selective PI3Kδ inhibitor. Based on encouraging clinical data from the phase Ib study (2021 ESMO, abstract# 8330), Amdiz is a designated breakthrough therapy in China for treatment of relapsed and refractory follicular lymphoma. Herein, we report the pre-clinical anti-tumor activity of Amdiz. Methods: Kinase activity was measured by Transcreener™ Fluorescence Polarization assay. The kinome selectivity profile of Amdiz was evaluated in Eurofins KinaseProfiler™ panel containing 323 kinases. Cell based phosphorylation of AKT was determined by using Acumen Explorer system. CD63 expression in basophils was evaluated by FACS assay. Inhibitory effects of Amdiz alone or in combination with other agents on cell viability were investigated in a panel of B cell lymphoma cell lines by CellTiter-Glo luminescent or CCK-8 assay. The ability of Amdiz to inhibit the activation of B cells in animals was evaluated in naïve rats using an ex vivo assay, and B cell activation was determined by evaluating CD86 expression using FACS analysis. Human B cell lymphoma cell line derived xenograft models were used to determine the anti-tumor activity of Amdiz in combination with other agents such as rituximab, BTK inhibitor, and venetoclax. Results: Amdiz is a highly selective inhibitor of PI3Kδ, showing more than 250-fold selectivity over other PI3K isoforms and no significant inhibition over other 319 protein kinases at the concentration of 1 µM. Amdiz potently inhibited PI3Kδ in biochemical, cellular and human whole blood assays with IC50s ranging from 0.8-3 nM. Its inhibitory effects on cell viability were also evaluated in a panel of B -cell lymphoma cell lines. Results showed that Amdiz potently inhibited cell survival with IC50s from 0.005 to 5 μM. Amdiz showed a long-lasting and strong inhibition on B cell activation in a rat pharmacodynamics (PD) study at a dose as low as 0.1 mg/kg. Moreover, Amdiz significantly improved anti-tumor activity of standard-of-care agents as well as targeted agents in multiple B-cell lymphoma models both in vitro and in vivo. Conclusion: Amdiz is a highly potent and selective PI3Kδ inhibitor with strong activity against B-cell lymphoma in pre-clinical studies, supporting clinical evaluation as either a single agent or in combination with other therapeutic agents for the treatment of B-cell malignancies. Citation Format: Jia Hu, Jianhan Wang, Xiaoming Dai, Jianlin He, Junqing Liang, Ying Yu, Juntao Yu, Na Yang, Linfang Wang, Yu Cai, Xiong Li, Weiguo Qing, Yongxin Ren, Weiguo Su. Amdizalisib (HMPL-689), a highly selective PI3Kδ inhibitor, exhibits potent anti-tumor activity in pre-clinical B-cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5454.

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

Abstract Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin lymphoma (NHL) and the most common form of adult lymphoma. Despite efforts to develop new therapies, the standard of care for DLBCL remains chemotherapy based. The current standard of care is comprised of a regimen of 4 drugs (cyclophosphamide, doxorubicin, vincristine, and prednisone) combined with immunotherapy using a chimeric mAb against the protein CD20 (rituximab), R-CHOP. Unfortunately only 50 to 60% of patients with DLBCL achieve and maintain complete remission after first-line therapy and 40 to 50 % relapse or have refractory disease. In addition, the average age at the time of diagnosis for DLBCL is 64 which makes intensive chemotherapy challenging. Therefore, the development of new treatment options is critical. We explored preclinical activity of AZD2811 nanoparticle, a potent and selective inhibitor of Aurora B kinase combined with venetoclax, an inhibitor of BCL2 currently used as standard of care in treatment of Haematological cancers, in B-Cell non-Hodgkin lymphoma models. Twenty five B-cell NHL cell lines including 11 Diffuse Large B-cell Lymphoma (DLBCL) cell lines were exposed to a 7 x 7 matrix of AZD2811 and venetoclax for 72 hrs. Cell viability was assessed using Celltiter-glo. Strong combination activity (HSA &amp;gt; 0.1 and combination Emax &amp;gt; 0.5) was observed in 6 cell lines including 2 DLBCL cell lines (WSU-DLCL2 and KARPAS_422). Moreover, pre-treatment with pan caspase inhibitor Q-VD-OPH, increased cell viability in cells treated with the combination. Next we explored the significant gene expression and mutation biomarkers (effect size &amp;gt; 1, p &amp;lt; 0.05) for this combination associated with Bliss and combination Emax in B-cell NHL cell lines. We found downregulation of 4 genes (FLT4, TIE1, PRKN) and mutations in 2 genes (EP300 and PTEN) to be associated with Bliss score and upregulation of one gene (FGR) associated with combination Emax. In vivo, antitumor activity of AZD2811 combined with venetoclax was assessed in mice bearing WSU-DLCL2luc xenografts. Once weekly intravenous administration of 25 mg/kg AZD2811 resulted in a statistically significant tumor growth inhibition (TGI) of 74%, while daily oral gavage of 100 mg/kg venetoclax resulted in 49% TGI but failed to reach statistical significance. While both monotherapies were unable to prevent progressive tumor growth, a combination dosing regimen of AZD2811 and venetoclax drove progressive tumor regression resulting in statistically significant complete regression (98% regression) by the third week of dosing. These results suggest combining AZD2811 with venetoclax has potential to be active in DLBCL patients. Citation Format: Azadeh Cheraghchi-Bashi, Brandon Willis, Kevin. Mongeon, Jacob Gordon, Krishna Bulusu, Elizabeth Coker, Patricia Jaaks, Mathew Garnett, Jerome Mettetal. Activity and tolerability of combination of venetoclax with the Aurora kinase B Inhibitor AZD2811 in preclinical diffuse large B-cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6303.

Abstract 2654: Anti-tumor activity of irreversible menin inhibitor, BMF-219, in high-grade B-cell lymphoma and multiple myeloma preclinical models

Abstract Double/Triple Hit Lymphoma (DHL/THL) and Double Expressor Lymphoma (DEL) are high-grade B-cell lymphomas (HGBCL) that exhibit low responses to standard therapeutic regimens resulting in poor prognosis. DHL harbor translocations in MYC and BCL2 or BCL6, THL contain translocations in MYC/BCL2/BCL6, and DEL are characterized by high expression of MYC and BCL2. Menin is a scaffold protein that drives oncogenic function through its regulation of genes such as HOXA9, with distinct effects on transcription that are directed by various cofactors. A recent study reported that knockdown of HOXA9 resulted in marked growth inhibition of multiple myeloma (MM) cells (Chapman et al., 2017). We previously reported the ability of irreversible menin inhibitor, BMF-219, to modulate MYC expression and exhibit high potency against DHL DLBCL preclinical models. Moreover, we demonstrated that MYC and its co-factor MAX, regulate differential gene expression in BMF-219-treated leukemia cells. Here, we demonstrate the anti-tumor activity of BMF-219 in HGBCL and MM preclinical models harboring various mutational backgrounds. BMF-219 exhibited high potency against THL and DEL cell lines, with IC50 values of 0.27 μM and 0.37 μM, respectively. Both models achieved &amp;gt;90% growth inhibition with single-agent treatment. Notably, BMF-219 was multi-fold more potent and exerted dramatically greater growth inhibition compared to clinical reversible menin inhibitors in all DLBCL cell lines tested, including an expanded panel of DHL cell lines. In ex vivo studies, an R-CHOP refractory THL patient sample and an R-EPOCH refractory MYC-amplified DLBCL patient sample were highly sensitive to BMF-219 treatment, with IC50 values of 0.15 μM and 0.2 μM, respectively, and demonstrated complete growth inhibition at 1 μM exposure. In contrast, two clinical reversible menin inhibitors demonstrated much lower potency (IC50 = 0.96 μM and 6.3 μM in the MYC-amplified model, and IC50 = 5.63 μM and not calculable for the second reversible inhibitor in the THL model). MM cell lines harboring mutations in TP53, KRAS and NRAS were all sensitive to BMF-219 with IC50 values in the range of 0.25 μM to 0.5 μM and achieved 100% growth inhibition at 1 μM exposure. Combination treatments of BMF-219 with standard-of-care agents in MM cell lines were also tested. Notably, BMF-219 demonstrated single-agent efficacy (IC50 values between 0.1 μM and 0.3 μM) against a panel of newly diagnosed and R/R ex vivo MM samples, including a p53-deleted clinical profile. Collectively, our data demonstrate the novel and robust anti-tumor activity of BMF-219 in HGBCL and MM preclinical models that represent categories of high unmet need. BMF-219 exhibits multi-fold higher potency and complete growth inhibition in these preclinical models compared to clinical reversible menin inhibitors, demonstrating its unique anti-tumor potential in these cancers. Citation Format: Priyanka Somanath, Daniel Lu, Brian Law, James T. Palmer, Taisei Kinoshita, Mini Balakrishnan, Thomas Butler. Anti-tumor activity of irreversible menin inhibitor, BMF-219, in high-grade B-cell lymphoma and multiple myeloma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2654.

Preclinical activity of irreversible Menin inhibitor, BMF-219, in chronic lymphocytic leukemia.

7541 Background: Menin is a scaffold protein that drives oncogenic function through transcriptional modulation directed by its various cofactors. A previous report demonstrated that menin regulates a distinct set of gene targets independent of its function with the MLL proteins in hematopoiesis and is essential for B-cell maturation (Li et al. Blood.2013;122(12):2039-46.). Chronic Lymphocytic Leukemia (CLL) is a disease of malignant B lymphocytes, for which standard-of-care agents are generally well tolerated; however, CLL patients with certain genetic backgrounds demonstrate inferior outcomes to these regimens. A major driving feature of CLL is overexpression of the anti-apoptotic marker, BCL2. We previously reported the ability of BMF-219, a selective, irreversible menin inhibitor, to downregulate the expression of BCL2 in acute leukemia cells. Additionally, we have reported the synergy of BCL2-targeted agent, venetoclax, with BMF-219 in potent cell killing of diffuse large B-cell lymphoma (DLBCL) preclinical models, prompting our exploration of BMF-219 activity in CLL. Here, we provide the first preclinical evidence for menin as a therapeutic target in CLL, by demonstrating high potency of BMF-219 against a diverse collection of CLL patient specimens. Methods: A comprehensive panel of CLL samples isolated from patients with Rai Stage 1 to 3 disease, including relapsed or refractory disease, were cultured ex vivo in the presence of BMF-219 or clinical reversible menin inhibitors to assess the antileukemic activity of the compounds. Samples were analyzed for differential gene expression of select targets in response to treatment. Results: BMF-219 demonstrated high potency, achieving &gt; 98% cell lethality at 1 µM exposure in all patient samples tested, with IC50 values in the range of 0.1 to 0.38 µM. Specimens isolated from patients with clinical profiles of high-risk genetic backgrounds associated with inferior outcomes to standard therapy, such as mutations in TP53 and NOTCH1, and chromosomal aberrations such as del(13q), trisomy 12 and complex karyotype, exhibited high sensitivity to BMF-219 treatment. BMF-219 was also highly effective against patient samples with clinical profiles of resistance to bendamustine, ibrutinib and venetoclax therapy. In comparison, clinical reversible menin inhibitors demonstrated no significant activity across all patient samples tested, with incalculable IC50 values and &lt; 15% reduction in cell viability at 1 µM exposure. Expression of select target genes in treated CLL cell lines was explored and will be reported. Conclusions: Collectively, our data demonstrate the potent preclinical activity of BMF-219 against CLL patient specimens harboring various mutational and cytogenetic backgrounds, including categories of high-risk. These data highlight the unique potential of irreversible menin inhibition as a novel therapeutic option for patients with CLL.

89Zr-ImmunoPET Shows Therapeutic Efficacy of Anti-CD20-IFNα Fusion Protein in a Murine B-cell Lymphoma Model.

Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved antitumor potency of anti-CD20-IFNα fusion proteins in preclinical mouse models of B-cell lymphoma. Although tumor targeting is mediated by the antibody part of the fusion protein, the cytokine component might strongly influence biodistribution and pharmacokinetics, as a result of its affinity, size, valency, and receptor distribution. Here, we used immunoPET to study the in vivo biodistribution and tumor targeting of the anti-CD20 rituximab-murine IFNα1 fusion protein (Rit-mIFNα) and compared it with the parental mAb (rituximab, Rit). Rit-mIFNα and Rit were radiolabeled with zirconium-89 (89Zr, t1/2 78.4 hours) and injected into C3H mice bearing syngeneic B-cell lymphomas (38C13-hCD20). Dynamic [(2 hours post injection (p.i.)] and static (4, 24, and 72 hours) PET scans were acquired. Ex vivo biodistribution was performed after the final scan. Both 89Zr-Rit-mIFNα and 89Zr-Rit specifically target hCD20-expressing B-cell lymphoma in vivo. 89Zr-Rit-mIFNα showed specific uptake in tumors (7.6 ± 1.0 %ID/g at 75 hours p.i.), which was significantly lower than 89Zr-Rit (38.4 ± 9.9 %ID/g, P < 0.0001). ImmunoPET studies also revealed differences in the biodistribution, 89Zr-Rit-mIFNα showed rapid blood clearance and high accumulation in the liver compared with 89Zr-Rit. Importantly, immunoPET clearly revealed a therapeutic effect of the single 89Zr-Rit-mIFNα dose, resulting in smaller tumors and fewer lymph node metastases compared with mice receiving 89Zr-Rit. Mice receiving 89Zr-Rit-mIFNα had enlarged spleens, suggesting that systemic immune activation contributes to therapeutic efficacy in addition to the direct antitumoral activity of IFNα. In conclusion, immunoPET allows the noninvasive tracking and quantification of the antibody-cytokine fusion protein and helps understand the in vivo behavior and therapeutic efficacy.

Clinical Studies on Cytokine-Induced Killer Cells: Lessons from Lymphoma Trials.

Simple SummaryLymphoma is a heterogeneous group of neoplasms including over 70 different subtypes. Its biological characteristic of deriving from lymphoid tissues makes it ideal for immunotherapy. In this paper, we provide insights into lymphoma-specific clinical trials based on cytokine-induced killer (CIK) cell therapy. We also reviewed pre-clinical lymphoma models where CIK cells have been used along with other synergetic tumor-targeting immune modules to improve their therapeutic potential. From a broader perspective, we will highlight that CIK cell therapy has potential, and in this rapidly evolving landscape of cancer therapies its optimization (as a personalized therapeutic approach) will be beneficial in lymphomas.Cancer is a complex disease where resistance to therapies and relapses often pose a serious clinical challenge. The scenario is even more complicated when the cancer type itself is heterogeneous in nature, e.g., lymphoma, a cancer of the lymphocytes which constitutes more than 70 different subtypes. Indeed, the treatment options continue to expand in lymphomas. Herein, we provide insights into lymphoma-specific clinical trials based on cytokine-induced killer (CIK) cell therapy and other pre-clinical lymphoma models where CIK cells have been used along with other synergetic tumor-targeting immune modules to improve their therapeutic potential. From a broader perspective, we will highlight that CIK cell therapy has potential, and in this rapidly evolving landscape of cancer therapies its optimization (as a personalized therapeutic approach) will be beneficial in lymphomas.

Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL).Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand).Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays.Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability.Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo.By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro.Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds.Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. DisclosuresSims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.

Combination of Loncastuximab Tesirine and Polatuzumab Vedotin Shows Increased Anti-Tumor Activity in Pre-Clinical Models of Non-Hodgkin Lymphoma

Loncastuximab tesirine-lpyl (formerly ADCT-402) is an antibody-drug conjugate (ADC) comprising a humanised anti-CD19 monoclonal antibody conjugated to the pyrrolobenzodiazepine (PBD) dimer-based payload tesirine. Once bound to CD19 on the cell membrane, loncastuximab tesirine is rapidly internalised and the released PBD dimer warhead causes interstrand DNA crosslinks which ultimately trigger cell death. Pre-clinically, loncastuximab tesirine has shown potent and specific anti-tumor activity in lymphoma models both as single agent and in combination with other approved drugs, like venetoclax, idelalisib and bendamustine (Zammarchi, Corbett et al. 2018, Tarantelli, Spriano et al. 2019). Loncastuximab tesirine has been recently approved by the United States Food and Drug Administration (FDA) for the treatment of relapsed or refractory (r/r) diffuse large B-cell lymphoma (DLBCL) and it is currently being tested in multiple clinical trials, either as monotherapy or in combination with other anti-lymphoma drugs.Polatuzumab vedotin is an ADC composed of a humanized anti-CD79b monoclonal antibody conjugated to monomethyl auristatin E (vcMMAE) and it is approved by the FDA for treatment of r/r DLBCL when used in combination with bendamustine and rituximab.Here, we investigated the in vitro and in vivo anti-tumor activity of loncastuximab tesirine combined with polatuzumab vedotin in pre-clinical models of non-Hodgkin lymphoma (NHL).In vitro, the combination of loncastuximab tesirine and polatuzumab vedotin was tested in three human-derived, CD19 and CD79b-positive NHL cell lines (WSU-DLCL2, TMD8 and Ramos) and it resulted in synergistic (TMD8 and Ramos) and additive (WSU-DLCL2) activity, as assessed by the Chou-Talalay method.Quantification of cell viability (propidium iodide [PI]-negative and Annexin V-negative) and early/late apoptosis (Annexin V-positive and PI-negative/ Annexin V-positive and-PI positive) on TMD8 and Ramos cells treated with loncastuximab tesirine, polatuzumab vedotin or the combination of the two agents showed a significant reduction of viable cells accompanied by an increase in apoptotic cells in the combination setting compared to the single agents.In vivo, loncastuximab tesirine was tested either alone (0.25 or 0.5 mg/kg, single dose) or in combination with polatuzumab vedotin (1 mg/kg, single dose) in the WSU-DLCL2 xenograft model. At the highest dose of loncastuximab tesirine, combination with polatuzumab vedotin resulted in improved anti-tumor activity and superior response rate compared to the 2 agents in monotherapy. All treatment regimens were well tolerated by the mice, as assessed by body weight measurements and frequent observation for signs of treatment-related side effects.In conclusion, the combination of loncastuximab tesirine and polatuzumab vedotin resulted in improved anti-tumor activity both in vitro and in vivo in lymphoma preclinical models and it was well tolerated. Altogether, these novel pre-clinical data warrant translation of the combination of loncastuximab tesirine and polatuzumab vedotin into the clinic for the treatment of NHL. DisclosuresSachini: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Jabeen: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. van Berkel: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Zammarchi: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

CC-99282 is a Novel Cereblon (CRBN) E3 Ligase Modulator (CELMoD) Agent with Enhanced Tumoricidal Activity in Preclinical Models of Lymphoma

CC-99282 is a novel, oral CELMoD ® agent currently under investigation in phase 1 clinical studies in patients with relapsed or refractory (R/R) non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Mechanistically, CC-99282 interacts with the CRL4 CRBN E3 ubiquitin ligase substrate receptor CRBN to induce recruitment and ubiquitin-mediated proteasomal degradation of transcription factors Ikaros and Aiolos. The design intent for CC-99282 included efficient absorption, deep tissue distribution, and prolonged exposure to optimize activity in bulky lymphoma lesions. Recently, we reported that CC-99282 shows potent antitumor activity in different preclinical models of diffuse large B cell lymphoma (DLBCL; Lopez-Girona, et al. Hematol Oncol. 2021). Here, we provide an expanded analysis of CC-99282 activity as a monotherapy, as well as examine its synergistic activity with anti-CD20 antibody treatment, in preclinical models of NHL including DLBCL and follicular lymphoma (FL).Compared with existing agents targeting Ikaros/Aiolos that show activity in hematologic malignancies, such as lenalidomide, avadomide, and iberdomide (CC-220), CC-99282 induced a more rapid, deep, and sustained degradation of Ikaros/Aiolos, causing derepression of cyclin-dependent kinase (CDK) inhibitors and interferon-stimulated genes (IRF7, IFIT3, and DDX58), and the reduction of the highly critical oncogenic factors c-Myc and IRF4. These molecular changes were followed by potent, 10- to 100-fold enhanced, autonomous cell killing and induction of apoptosis (Figure). Our results show that these effects were independent of the cell of origin (activated B cell [ABC; TMD8 cell line], germinal center B cell [GCB; WSU-DLCL2 cell line], or primary mediastinal B cell lymphoma [PMBL] subtypes of DLBCL) or presence of high-risk chromosomal translocations (MYC, BCL2, and/or BCL6), as observed in a panel of 36 lymphoma cell lines that included DLBCL and FL cell lines. In vivo, CC-99282 demonstrated robust tissue distribution that favored target tissues and exhibited antitumor activity resulting in improved tumor regression and tumor-free animals in several lymphoma xenograft models, including an intracranial xenograft model. This strong antitumor activity was observed using various continuous and intermittent dosing paradigms.The potent, direct autonomous cell-killing activity of CC-99282 was augmented when CC-99282 was combined with the anti-CD20 antibody rituximab. In vitro combination studies of CC-99282 with rituximab in lymphoma cell lines demonstrated enhanced cell killing by human natural killer (NK) cells, macrophage-mediated phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In FL and DLBCL cell lines, we showed that the combination of CC-99282 with rituximab resulted in increases in both NK-mediated ADCC and macrophage-mediated ADCP of up to 20% compared with rituximab treatment alone. In vivo, combination treatment with CC-99282 and rituximab induced dose-dependent tumor growth inhibition in WSU-DLCL2 and RL (FL) xenograft models. In the WSU-DLCL2 model, CC-99282 (1 mg/kg) or rituximab (10 mg/kg) monotherapy resulted in modest tumor growth inhibition, whereas the combination of CC-99282 (1 mg/kg) and rituximab (10 mg/kg) resulted in tumor regression in 100% of animals. Similar results were obtained in FL xenograft models using the RL cell line, where combinations of CC-99282 (1 mg/kg) with rituximab (25 mg/kg) induced complete tumor regression in 100% of animals.In conclusion, CC-99282 is a novel CELMoD agent with an improved substrate degradation profile compared with existing Ikaros/Aiolos-degrading agents. CC-99282 demonstrated enhanced antiproliferative and apoptotic activities across a broad range of lymphoma cells and a robust distribution profile that favors target tissues such as lymphoid organs. In addition, CC-99282 acts synergistically in combination with anti-CD20 monoclonal antibody treatment. Collectively, these data support the clinical investigation of CC-99282 as monotherapy and in combination with rituximab in patients with R/R NHL. [Display omitted] DisclosuresCarrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Groocock: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Janardhanan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jankeel: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Galasso: Ryan Galasso: Current Employment, Current equity holder in publicly-traded company. Guarinos: Bristol Myers Squibb: Current Employment. Narla: Bristol Myers Squibb: Current Employment. Groza: Bristol Myers Squibb: Current Employment. Leisten: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Rolfe: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lopez-Girona: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

A Phase I Trial of PI3Kαδ Inhibitor Copanlisib in Combination with Nivolumab in Patients with Richter's Transformation (RT) or Transformed Non-Hodgkin Lymphoma (tNHL)

Background. Despite advances in targeted and cellular therapy, outcomes among patients with RT and tNHL remain dismal. Copanlisib (COPA) is a selective, small molecule, PI3K inhibitor which preferentially targets the p110αδ isoforms . COPA has shown clinical efficacy in NHL and is an approved therapy for follicular lymphoma (FL). Nivolumab is a PD-1 antagonist which has demonstrated activity in Hodgkin lymphoma as well as NHL. Furthermore, combined targeting of PI3K and PD-1 has demonstrated synergy in pre-clinical lymphoma models. Here we report initial results of a phase 1 study of COPA in combination with nivolumab in patients with R/R RT or tNHL (NCT03884998).Methods. This ongoing multicenter, open-label, phase I, investigator-sponsored study is enrolling patients with RT or tNHL, age ≥18 years, whose disease had relapsed or was refractory to ≥1 prior line of therapy. The phase I portion of the study followed a standard 3+3 design with three planned dose levels of COPA administered IV (dose level [DL]1 - 45 mg on days 1, 8 and 15; DL2 - 60 mg on days 1, 8 and 15; DL “-1” - 45 mg on days 1 and 15 of a 28-day cycle). Nivolumab 240 mg was given IV on days 1 and 15. Patients received up to 24 cycles of therapy. The primary study objective was to evaluate the maximum tolerated dose (MTD) of the combination; secondary objectives included preliminary measures of efficacy by Lugano criteria including PET/CT scans. Exploratory objectives included pharmacodynamic endpoints. Dose limiting toxicities (DLT) were defined as grade ≥4 hematologic > 7 days or grade ≥3 non-hematologic toxicities.Results. Of the eleven patients enrolled, 8 had RT and 3 had transformed FL (tFL); 91% (10/11) were men. Median age was 70 (43-77) years, 82% (9/11) had an ECOG performance status ≤1. Patients had received a median of 3 prior lines of therapy (range, 1-8), including three patients (1 RT, 2 FL) who had undergone CAR T cell therapy. Eight patients were treated at DL1 (45 mg COPA), of which 2 patients did not complete the DLT period due to rapidly progressive disease and were replaced. No DLTs were observed at this dose level. At DL2 (60 mg COPA), three DLTs (grade 4 febrile neutropenia and grade 4 thrombocytopenia in another patient) were observed among the three patients treated. Therefore, the MTD and recommended phase 2 dose (RP2D) of COPA in combination with nivolumab was determined to be 45 mg on days 1, 8, 15. The most common treatment-related adverse events (AEs, any grade) were anemia and neutropenia (Table 1). Five pts discontinued therapy due to progressive disease; one due to intercurrent infection (coccidiomycosis, unrelated to study drugs), two due to DLTs noted above. Three patients remain on treatment. Among the seven efficacy-evaluable patients who completed at least one cycle of therapy, median time on treatment was 2 months (range, 2-9 months). Of these, four patients achieved a response per the investigators' assessment (tFL: 2 partial responses; RT: 1 complete response and 1 partial response). Efficacy assessment is ongoing and will be reported at the meeting.Discussion. We identified COPA 45 mg IV on days 1, 8 and 15 as the MTD/RP2D in combination with nivolumab in patients with R/R RT and tFL. This combination was well-tolerated at the MTD/RP2D, and the study is currently enrolling patients in an expansion cohort to further characterize the efficacy of this regimen. [Display omitted] DisclosuresShouse: Beigene Pharmaceuticals: Honoraria; Kite Pharmaceuticals: Speakers Bureau. Siddiqi: Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Juno therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BeiGene: Other: DSM Member, Speakers Bureau; PCYC: Speakers Bureau; Jannsen: Speakers Bureau; Dava Oncology: Honoraria; ResearchToPractice: Honoraria. Popplewell: Pfizer: Other: Travel; Novartis: Other: Travel; Hoffman La Roche: Other: Food. Kittai: Janssen: Consultancy; Bristol-Meyers Squibb: Consultancy; Abbvie: Consultancy. Davids: MEI Pharma: Consultancy, Research Funding; Surface Oncology: Research Funding; AbbVie: Consultancy; BeiGene: Consultancy; Merck: Consultancy; Ascentage Pharma: Consultancy, Research Funding; Eli Lilly and Company: Consultancy; BMS: Consultancy, Research Funding; Janssen: Consultancy; Celgene: Consultancy; Astra-Zeneca: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Research to Practice: Consultancy; Verastem: Consultancy, Research Funding; Takeda: Consultancy; MEI Pharma: Consultancy; TG Therapeutics: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Genentech: Consultancy, Research Funding. Danilov: Genentech: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria; Takeda Oncology: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Gilead Sciences: Research Funding; Pharmacyclics: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; TG Therapeutics: Consultancy, Research Funding.

Clinical and Biological Effects of Combined CD27 and CD20 Antibody Therapy in Relapsed/Refractory B-Cell Lymphoma: The Riva Trial

Clinical and Biological Effects of Combined CD27 and CD20 Antibody Therapy in Relapsed/Refractory B-Cell Lymphoma: The Riva Trial

CCS1477, a Novel p300/CBP Bromodomain Inhibitor, Enhances Efficacy of Azacitidine and Venetoclax in Pre-Clinical Models of Acute Myeloid Leukaemia and Lymphoma

E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferases that co-activate key oncogenes such as MYC and IRF4 which are relevant in a number of haematological malignancies. Here we describe the effects of CCS1477, a new orally available inhibitor of the p300/CBP bromodomains, given alone or in combination with azactidine or venetoclax in pre-clinical models of AML and B-cell lymphoma.As a monotherapy, daily oral dosing with CCS1477 (5, 10 and 20mg/kg) caused a significant dose-dependent reduction in tumour growth in a MOLM-16 xenograft model of AML, with regressions observed at the highest and well tolerated dose of 20mg/kg/qd. The inhibition of tumour growth during drug treatment was accompanied by a significant reduction in tumour expression of MYC by qPCR. In murine retroviral transduction and transplantation models of human AML initiated by either MLL-AF10 or MLL-AF9, daily monotherapy by oral gavage with CCS1477 at 30mg/kg for 42 days, caused a highly significant prolongation of survival. All seven control treated mice died of AML within 160 days whereas only one of seven CCS1477 treated mice had died in each model by 300 days.In the MOLM-16 xenograft model, a sub-maximal dose of CCS1477 (10mg/kg) demonstrated superior tumour growth inhibition by comparison with azacitidine (0.5mg/kg i.p) and with significant combination benefit when these two agents were combined. In this same model venetoclax (50 or 100 mg/kg/qd oral) had no effect on tumour growth, indicating that MOLM-16 tumours are intrinsically resistant. CCS1477 dosed daily at 10mg/kg restored sensitivity to venetoclax as evidenced by significantly greater tumour growth inhibition when compared with CCS1477 given alone. In a venetoclax-sensitive xenograft model of AML (MV-4-11), CCS1477 (10mg/kg) and venetoclax (100mg/kg) resulted in a similar and partial reduction in tumour growth over a 21 day dosing period. When venetoclax and CCS1477 were combined there was a significant combination benefit compared to either drug given alone. The DOHH-2 xenograft model of B-cell lymphoma was also sensitive to venetoclax (50mg/kg) and with a similar impact on tumour growth compared with CCS1477 (10mg/kg). In this model the combination of CCS1477 and venetoclax was more profound and caused complete tumour stasis during the dosing period.These data support the clinical testing of CCS1477 in combination with azacitidine and/or venetoclax in AML and B-cell lymphomas. CCS1477 is currently in Phase I/II clinical trials in AML, Non-Hodgkin lymphoma (including B-cell lymphoma) and multiple myeloma. (NCT04068597). DisclosuresBrooks: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Knurowski: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Hughes: CellCentric Ltd: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Clegg: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. West: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Pegg: CellCentric Ltd: Current Employment, Current holder of individual stocks in a privately-held company. Somervaille: Novartis: Consultancy, Honoraria.

Combination Benefit of Capivasertib and Venetoclax in Preclinical Models of Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy among adults and despite approximately 65% of patients with DLBCL being cured with RCHOP therapy, nonresponsive and relapsed patients have inadequate treatment options, highlighting the importance for innovative treatment regimens. Blockade of B-cell receptor (BCR) downstream signaling components with various targeted agents is emerging as a clinically tractable treatment strategy across multiple B-cell malignancies. Protein Kinase B (AKT) signaling downstream of the BCR complex has been shown to be a central node in germinal center B-cell (GCB) DLBCL and the potent, selective inhibitor of AKT1, AKT2, AKT3, capivasertib, currently being evaluated in multiple clinical trials by targeting AKT-driven solid cancers, has been shown to induce apoptosis in a subset of GCB-DLBCL cell lines and cause tumor stasis in xenograft mouse models (Erdman et al., 2017). Since the monotherapy capivasertib responses in GCB DLBCL models are partial and lack durability, we hypothesized a combination approach could deliver even greater therapeutic benefit. To identify optimal partners, we conducted a capivasertib centric in vitro combination screen with specific with BH3 family members across a panel of 15 DLBCL cell lines, which revealed a synergistically active combination with the BCL2 inhibitor, venetoclax which is currently being evaluated in DLBCL. The activity was specifically enhanced in cell lines of the GCB subtype, with 4 PTEN del and 2 PTEN wt cell line models showing combination benefit. To determine the ability of this combination to drive stronger and durable responses, we assessed capivasertib and ventoclax activity in xenograft mouse models using two GCB-DLBCL cell line lines, SUDHL4 (PTEN wt) and WSU-DLCL2 (PTEN del). Oral administration of either monotherapy capivasertib (130 mg/kg BID, 4-day on/3-day off) or venetoclax (100 mg/kg QD) provided partial tumor growth inhibition (capivasertib TGI = 74% in SUDHL4 and 29% in WSU-DLCL2, and venetoclax TGI = 46% in SUDHL4 and 0% in WSU-DLCL2), whereas the combination of capivasertib and venetoclax both on a 4-day on/3-day off schedule produced complete tumor regression (100% regression) in both xenograft GCB cell line models during the dosing period. Notably, in both xenograft models all mice (5/5 per model) remained tumor free for at least 30 days following dosing cessation demonstrating high durability of response for the combination. Additionally, this combination is currently being evaluated in clinically relevant GCB and non-GCB PDX mouse models. Taken together, our results provide preclinical evidence for the rational combination of AKT and BCL-2 blockade with capivasertib and venetoclax respectively in patients with relapsed/refractory GCB-DLBCL. DisclosuresWillis: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Neveras: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Dry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mongeon: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Rosen: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mettetal: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Barry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options.

A Novel Antibody Drug Conjugate (ADC) Targeting Cell Surface Heat Shock Protein 70 (csHSP70) Is Active Against Pre-Clinical Models of Peripheral T-Cell Lymphoma (PTCL)

A Novel Antibody Drug Conjugate (ADC) Targeting Cell Surface Heat Shock Protein 70 (csHSP70) Is Active Against Pre-Clinical Models of Peripheral T-Cell Lymphoma (PTCL)

NL-201, a De Novo Agonist of IL-2 and IL-15 Receptors, Demonstrates Synergistic Antitumor Activity with Anti-PD-1 Checkpoint Inhibitor Therapy in a Preclinical Non-Hodgkin Lymphoma Model

NL-201 is a potent, selective, and long-acting computationally designed alpha-independent agonist of the IL-2 and IL-15 receptors that is being developed as an immunotherapy for cancer. NL-201 binds to the beta and gamma subunits, selectively stimulating dose-dependent expansion and tumor infiltration of cytotoxic CD8+ T cells and natural killer (NK) cells, thereby enhancing the immune response to the tumor. Absence of binding to the IL-2 alpha subunit reduces the undesirable effects of traditional IL-2 therapies, such as vascular leak syndrome and expansion of immunosuppressive regulatory T cells. In this abstract, we demonstrate that NL-201, alone or in combination, demonstrates robust antitumor activity in preclinical models of non-Hodgkin lymphoma (NHL).We have previously demonstrated that NL-201 has marked antitumor activity in multiple syngeneic tumor models, including the A20 lymphoma model. These observations in lymphoma have been extended to explore the effects of NL-201 in combination with anti-mPD-1 checkpoint inhibitor therapy in vivo. In this model, NL-201 and anti-mPD-1 demonstrated tumor growth inhibition and increased median survival (21 days each vs 17 days as observed in control) when given alone. In combination, NL-201 and anti-mPD-1 resulted in increased antitumor activity and significant prolongation of survival (>51 days). We have also demonstrated that NL-201 does not directly induce signaling or cell death in B cell−derived NHL, suggesting that the observed antitumor activity is due to activation of non-malignant host immune cells.Additional in vitro and in vivo NHL models are being tested to enhance understanding of the interaction between NL-201 and other approved therapies within the hematopoietic tumor microenvironment. These data will be used to design future clinical trials of NL-201 in novel regimens to treat hematological malignancies. DisclosuresHuard: Neoleukin Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties. Tatalick: Neoleukin Therapeutics, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Independent paid nonclinical consultant for Neoleukin. Walkey: Neoleukin Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Swanson: Neoleukin Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.