CLL Cell Lines Research Articles

Abstract 3305: Golcadomide (CC-99282) is a novel CELMoD® agent with antiproliferative activity and combinatorial potential in disease models of chronic lymphocytic leukemia

Abstract Chronic Lymphocytic Leukemia (CLL) is an adult leukemia characterized by the accumulation of incompetent B lymphocytes expressing CD19, CD20, CD23, and CD5. Current therapies include chemotherapy and combination with targeted therapies such as ibrutinib (Bruton’s tyrosine kinase inhibitor), venetoclax (BCL2 inhibitor), and obinutuzumab (anti-CD20). The tumor microenvironment promotes CLL development and drug resistance. Golcadomide (GOLCA, CC-99282) is an oral cereblon E3 ligase modulator (CELMoD®) agent with immunomodulatory and tumor cell-autonomous activities under clinical investigation for R/R non-Hodgkin lymphomas and CLL/SLL. Here we investigated the effect of GOLCA on inhibiting proliferation and inducing apoptosis of CLL preclinical models by degrading Ikaros and Aiolos. We also studied its combination with other anti-CLL agents. A panel of 10 CLL cell lines and 15 primary CLL patient samples were used to assess efficacy of GOLCA alone or in combination with ibrutinib (Ibru), venetoclax (Ven), and obinutuzumab (Obi). Primary CLL cells were grown ex vivo in a co-culture system with CD40L expressing fibroblasts designed to mimic the CLL lymph node microenvironment. Substrate degradation, viability, cell cycle and immunophenotyping were assessed by flow cytometry. GOLCA showed potent in vitro antiproliferative activity on 6 out of 10 CLL cell lines, including those with high-risk features, with IC50 of 1-20 nM. It also inhibited CLL stimulated cell proliferation and induced apoptosis in all evaluated patient samples in the co-culture system, with low to sub-nanomolar IC50, independent of IGHV mutation status and other chromosomal characteristics. Cell-cycle analysis confirmed inhibition of proliferation in the 5 patient samples evaluated after 3 days of treatment with GOLCA, with a dose-dependent decrease in the fraction of cells in the S phase. GOLCA also demonstrated degradation of the proximal substrates Ikaros and Aiolos in tumor and T cells from CLL patient samples. In combination with Obi, Ven, and Ibru, GOLCA led to synergistic or additive tumor cell toxicity in most patient samples evaluated, suggesting potential clinical benefit of these targeted agent combinations. Additive tumor toxicity was observed in two out of four patient samples when GOLCA was combined with Ibru. We have previously demonstrated that GOLCA induces immune activation in T cells from healthy donor PBMCs. In co-cultures of CLL PBMCs and CD40L-expressing fibroblasts, GOLCA increased T cell numbers and activation characteristics. Ven inhibited this immune activation, while Obi or Ibru maintained the immune activation. Collectively, these findings suggest that GOLCA, alone or in combination with targeted agents, may produce clinical benefit in CLL patients. Citation Format: Antonia Lopez-Girona, Maria Dolores Jimenez-Nunez, Diego Sobradillo, Soraya Carrancio, Preethi Janardhanan, Gauri Deb, Lynda Groocock, Daniel Pierce, Neil Bence, Mark Rolfe. Golcadomide (CC-99282) is a novel CELMoD® agent with antiproliferative activity and combinatorial potential in disease models of chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3305.

Development and Testing of a Glycoengineered Anti-ROR1 Antibody with Enhanced Capacity for Directing Antibody-Dependent Cellular Cytotoxicity (ADCC) of Chronic Lymphocytic Leukemia Cells

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic surface antigen expressed on the neoplastic cells of patients with chronic lymphocytic leukemia (CLL) or other types of cancer, but not on virtually all normal postpartum tissues (Fukuda T., et al., PNAS, 105:3047, 2008). Zilovertamab (also known as UC-961 or cirmtuzumab) is a humanized anti-ROR1 monoclonal antibody (mAb) currently under clinical investigation; this mAb is capable of blocking ROR1-signaling, which can contribute to cancer-cell migration, proliferation, survival, and self-renewal. We generated a glycoengineered form of zilovertamab (GE- zilovertamab) and examined whether it could mediate enhanced antibody-dependent cellular cytotoxicity (ADCC) against neoplastic cells that express ROR1. Initial studies used Jurkat-Lucia™ NFAT-CD16 cells (Invivogen, San Diego, CA, USA) which express high-affinity CD16A (FcγRIIIA, V158 allotype) as effector cells (EC), and the CLL-cell line MEC1, or MEC1 cells transduced to express ROR1 (MEC1-ROR1), as target cells (TC), which each also express CD20. Co-culture of Jurkat-Lucia cells for 6 hours with the anti-CD20 mAb rituximab and MEC1 or MEC1-ROR1 cells induced Jurkat-Lucia cells to express a luciferase reporter gene under the control of an ISG54 minimal promoter fused to six NFAT response elements; this endowed the Jurkat-Lucia cells with high luminescence activity that was not observed in co-cultures of EC and TC without added mAb. Neither zilovertamab nor GE-zilovertamab endowed Jurkat-Lucia cells with higher than background luminescence activity when co-cultured with MEC1 cells. However, GE-zilovertamab could induce high levels of luminescence activity in Jurkat-Lucia cells co-cultured with MEC1-ROR1 cells that was comparable to that of rituximab, and significantly greater than that of zilovertamab. We generated an orthogonal system with which to examine the relative ADCC activity of each of these mAb, using EC generated from the NK cell line, NK92, which we transduced with a lentivirus encoding a newly-generated high-affinity variant of CD16A (CD16v), which has been modified to block its cleavage by ADAM17. NK92 or NK92-CD16v were used as EC to effect cytolysis of Cr 51-labeled MEC1, MEC1-ROR1, or primary CLL cells in a chromium-release assay. Addition of rituximab to co-cultures of EC with Cr 51-labeled MEC1, MEC1-ROR1, or primary CLL caused mAb-dose-dependent ADCC of each of these TC by NK92-CD16v that was significantly greater than that by parental NK92 cells. Again, neither zilovertamab nor GE-zilovertamab could direct ADCC by NK92-CD16v or NK92 ADCC of MEC1 cells, which lack ROR1. However, GE-zilovertamab directed dose-dependent ADCC of MEC1-ROR1 or primary ROR1 + CLL cells by either NK92-CD16v or NK92 at levels comparable to that directed by rituximab for either EC population; such levels were significantly greater than that directed by zilovertamab. ROR1 + CLL cells harboring del(17p) or mutations in TP53 (del(17p)/m TP53) and/or that were resistant to targeted therapies (e.g., inhibitors of BTK or BCL2), were as susceptible to GE-zilovertamab-directed ADCC as were CLL cells without del(17p)/m TP53 from patients who had not had prior therapy. These data demonstrate that GE-zilovertamab can direct high-level ADCC lysis of ROR1-expressing neoplastic cells with greater activity than zilovertamab, encouraging development of clinical studies to evaluate GE-zilovertamab for therapy of patients with CLL or other ROR1-positive cancers.

Preclinical investigations of the efficacy of the glutaminase inhibitor CB-839 alone and in combinations in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells are metabolically flexible and adapt to modern anticancer treatments. Bruton tyrosine kinase (BTK) and B-cell lymphoma-2 (BCL-2) inhibitors have been widely used to treat CLL, but CLL cells become resistant to these treatments over time. CB-839 is a small-molecule glutaminase-1 (GLS-1) inhibitor that impairs glutamine use, disrupts downstream energy metabolism, and impedes the elimination of reactive oxygen species. To investigate the in vitro effects of CB-839 on CLL cells, we tested CB-839 alone and in combination with ibrutinib, venetoclax, or AZD-5991 on the HG-3 and MEC-1 CLL cell lines and on primary CLL lymphocytes. We found that CB-839 caused dose-dependent decreases in GLS-1 activity and glutathione synthesis. CB-839-treated cells also showed increased mitochondrial superoxide metabolism and impaired energy metabolism, which were reflected in decreases in the oxygen consumption rate and depletion of the adenosine triphosphate pool and led to the inhibition of cell proliferation. In the cell lines, CB-839 combined with venetoclax or AZD-5991, but not with ibrutinib, demonstrated synergism with an increased apoptosis rate and cell proliferation inhibition. In the primary lymphocytes, no significant effects of CB-839 alone or in combination with venetoclax, ibrutinib, or AZD-5991 were observed. Our findings suggest that CB-839 has limited efficacy in CLL treatment and shows limited synergy in combination with widely used CLL drugs.

APG115 Enhances the Sensitivity of Ibrutinib Via Promoting p53 to Target MCL-1 in Chronic Lymphocytic Leukemia

Introduction: APG115 is an orally active, selective, potent small molecule inhibitor of the MDM2-p53 interaction, which destabilizes MDM2-p53 complex and promotes p53 activation. Previous studies reported that APG115 promoted cell apoptosis in several types of cancer. However, its role and mechanism in the development of CLL are still unclear. Methods: Peripheral blood samples from de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University. The effects of APG115 on cell proliferation, apoptosis and cycle were detected by CCK8, Annexin V-PE/7AAD staining and PI/RNase staining respectively. Related protein expression of p53 pathway in CLL cell lines and primary cells were detected by Western blot. Protein interactions were verified by COIP. Immunofluorescence was applied to detect protein localization. Results: To study the role of APG115, we added APG115 into CLL cell lines and primary cells to select appropriate concentration according to IC50. The cells viability of gradually decreased along with the extension of time or concentration augment, which suggested that CLL cells were time-dose-dependent on APG115. In addition, the protein levels of MDM2, p53, PUMA and p21 increased under the treatment of APG115, indicating that APG115 relieved the inhibition of MDM2 on p53 and activated downstream apoptotic pathways. Meanwhile, the APG115 induced cell apoptotic elevation and cell cycle arrest in G0/G1 phase. Subsequently, aimed to explore whether APG115 affects the ibrutinib sensitivity in CLL cells, the cell viability of combination group under both APG115 and ibrutinib was obviously suppressed than groups with single drug intervention. Immunofluorescence images revealed that p53 promoted MCL-1 into cytoplasm under the action of APG115. However, on the basis of ibrutinib intervention, APG115 facilitated the degradation of MCL-1 by p53. Furthermore, the invasion range of CLL cells in the combination group was smaller than that in the single agent group, and the invasion range of CLL cells in the single agent group was lower than that in the control group in vivo(Figure A). After injection of medication, both the monotherapy group and the combination group were lower than the control group, but there was no significant difference between the monotherapy group and the combination group. The survival rate of combination group was higher than that of single agent group (Figure B). Immunohistochemistry of the dissected spleens of mice showed that the malignancy of the cells in the single agent group was lower than that in the control group, while that in the combination group was lower than that in the monotherapy group. Finally, ibrutinib-resistant cell line was established to study the effect of APG115 on chemoresistance. After adding APG115, it was found that the survival rate of drug-resistant strain was lower than that of sensitive strain. It was proved that p53 induced the ubiquitination process when combined with MCL-1 to reduce the inhibitory effect of apoptosis via CO-IP. After APG115 was added, the apoptosis rate of drug-resistant strains was higher than that of sensitive strains. When APG115 was added on the basis of ibrutinib, the difference in apoptosis rate of drug-resistant strains was greater than that of sensitive strains. Conclusions: In summary, this study first revealed anticancer effects of the novel MDM2-p53 inhibitor APG115 in CLL. Promisingly, APG115 is expected to be a potential agent to improve the effects of BTK inhibitor treatment in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effects of Glutaminase Inhibition on Venetoclax, Ibrutinib, and AZD-5991 Resistance in Chronic Lymphocytic Leukemia Cell Lines

Introduction: Bruton tyrosine kinase and BCL-2 inhibitors are widely used in CLL, but resistance problem is highly relevant. One of the resistance mechanisms may be metabolic rewiring followed by activation of oxidative phosphorylation. Glutamine plays a major role in cancer metabolism, and glutamine limitation rather than glucose contributes to lower oxygen uptake and mediates apoptosis of cancer cells. It was shown that glutamine metabolism changes contribute to venetoclax (Chen Z. et al, Blood, 2021; Guièze R. et al. Cancer Cell, 2019) and ibrutinib (Galicia-Vázquez G. et al. Frontiers in Oncology, 2018) resistance. We investigated in vitro effects of glutaminase-1 (GLS-1) enzyme inhibition and its potential synergistic effects with venetoclax, ibrutinib, and MCL-1 inhibitor AZD-5991 on resistant cell lines. CB-839 (telaglenastat) is a potent, reversible, and selective GLS-1 inhibitor with 24 nM IC50 for recombinant human mitochondrial glutaminase C. The compound was tested in the clinic for hematological malignancies and solid tumors. Methods: We have tested CB-839 alone or in combination with venetoclax, ibrutinib, and AZD-5991 on resistant HG-3 and MEC-1 CLL cell lines. Cells were incubated with 0.01 to 100 μM of CB-839 for 72 hours. The inhibition of glutamine, glutamate, and glutathione induced by CB-839 alone was measured using a luminescence assay. DCFDA/ H2DCFDA and MitoSOX Red stainings were utilized to evaluate cellular reactive oxygen species (Total ROS) and mitochondrial superoxide (MitoSOX) accumulation, respectively. Intracellular nucleotides including ATP level were measured using high-pressure liquid chromatography. For drug combinations, MTS assay and Annexin V/Propidium Iodide staining were used to assess cell proliferation and apoptosis, respectively. The combination index (CI) for synergy was calculated using Compusyn software. Results: HG3 and MEC1 cell lines were resistant to venetoclax, ibrutinib, and AZD-5991 single agents. The IC50 values for HG3 are 8.1 μM, 1.3 μM, 9.9 μM, and for MEC1 are 2.9 μM, 1.2 μM, and 1.9 μM for venetoclax, ibrutinib, and AZD-5991, respectively. In these cell lines, CB-839 caused a dose-dependent decrease in GLS-1 activity, measured as intracellular glutamate concentration, and inhibited glutathione synthesis (Figure 1A). CB-839 treated cells showed increased MitoSOX, but not Total ROS accumulation. However, as the endpoint effect, the decrease of cell proliferation rather than apoptosis was detected. CB-839 treatment caused a mild decrease in ATP pool in both cell lines. According to MTS assay, venetoclax demonstrated only the particular synergism with CB-839 (CI <1.0 at all effective doses (ED50, ED75, ED90, ED95) for MEC-1, and at ED50, ED75 for HG-3 cell line). In addition, CB-839 increased pro-apoptotic effect of venetoclax in both cell lines. Notably, that CB-839 did not improve any ibrutinib-mediated effects on cell proliferation and apoptosis in CLL cell lines. The most distinct synergism was observed in CB-839 and AZD-5991 combination group (CI <1.0 at all ED for MEC-1 and HG-3 cell lines) with 5- and 19.8-fold shifts in IC50 (Figure 1B). To extend these results, we are performing these combinations in primary CLL cells that are sensitive or resistant to targeted agents such as ibrutinib or venetoclax. Conclusions: Combinations of CB-839 with other agents targeting multiple mechanisms have the potential to overcome metabolic adaptation. Our findings suggest that CB-839 has limited efficacy in overcoming ibrutinib CLL resistance. However, the synergism with apoptosis inducers such as venetoclax and AZD-5991 require further investigations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pathological RANK Signaling in Combination with TCL1 Drives a Multiple Myeloma-like Disease in Mice

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL, play an important role in different cancer entities, including B cell malignancies (Renema et al, 2016). In diffuse large B cell lymphoma, recurrent RANK mutations were detected. When expressed in murine B cells, RANK(K240E) leads to the development of chronic lymphocytic leukemia (CLL; Alankus et al. 2021). In addition, the RANK/RANKL signaling pathway is also relevant for multiple myeloma (MM) niche formation and disease progression (Raje et al. 2019). To study the consequences of constitutive activation of RANK signaling in combination with the oncogene TCL1, we crossed our RANK(K240E) transgenic mouse model with the TCL1 mouse model to obtain Eµ-TCL1*RANK(K240E)*CD19Cre mice (TC-RK) mice and followed disease burden. When we analyzed the symptom-free survival, we observed that TC-RK mice have a reduced life span with a median survival of 30.1 weeks as compared to >50 weeks in TC mice. Flow cytometric analysis of terminally ill TC-RK mice revealed a strong accumulation of a RANK expressing, CD19-negative population in the spleen, bone marrow (BM) and liver of TC-RK mice, with up to 80-90% of lymphocytes harvested from these organs. Apart from their increased cell size, those RANK+CD19- cells showed strong expression of CD138 and reduced surface expression of B220, and no surface IgM and IgD compared to RANK+CD19+ cells, suggesting they have differentiated to plasma cells. Surprisingly, we observed low levels of CLL cells in the peripheral blood and the spleen, although RANK(K240E) and TCL1 alone induce a CLL-like disease in mice. However, similar to what is observed in human plasma cell disorders, including MM, serum electrophoresis of diseased TC-RK mice revealed sharp clonal spikes in the gamma-fraction, which were not detected in any RK- or TC-derived control serum. Furthermore, plasma cells of TC-RK mice readily engrafted and promoted monoclonal disease development in immunocompromised mice. This data strongly suggests that the cooperation of TCL1 with RANK(K240E) signaling promotes the transformation of plasma cells and implicates a direct role of B-cell intrinsic RANK signaling in MM pathogenesis. To gain insight into the pathogenesis of the MM-like disease in the TC-RK mice, we next examined whether RANK and/or TCL1 expression impacts the plasma cell differentiation capacity in vitro. Ex vivo stimulation with suboptimal LPS concentration revealed an increased differentiation into plasma cells in both, RK and TC-RK derived B cells, as compared to CD19Cre and TC mice. This indicates that active RANK signaling alone is sufficient to promote survival and plasma cell differentiation in vitro. In line, 6-week-old TC-RK and RK mice showed an increased plasma cell population in the spleen, but not in the BM, compared to TC and CD19Cre mice. At 20 weeks of age, only TC-RK mice showed a strong accumulation of plasma cells in the BM (29.9%±11.2, n≥4), spleen (29.7%±8.5, n≥4), and liver (11.7%±6, n≥4), suggesting that only the TC-RK expressing malignant cells disseminate and localize to extra-medullary sites at an advanced stage of disease, mimicking MM. We are currently analyzing bulk and single cell RNA sequencing data to gain insight into the pathways contributing to the disease pathogenesis in this model. To study the relevance of RANK expression in human myeloma cells, we analyzed RANK protein in levels in MM cell lines and observed comparable levels to CLL cell lines, that depend on RANKL for their in vivo progression. Denosumab, a monoclonal antibody blocking RANKL, is used in MM patients to reduce bone turnover. Notably, blocking RANKL in preclinical models reduces MM disease burden (Pearse et al. 2001) and anti-myeloma effects of denosumab have been discussed in MM patient subsets (Terpos et al. 2021). We are therefore currently testing the effects of anti-RANKL treatment on MM cells and evaluating, whether denosumab treatment sensitizes MM cells to other therapies. Taken together, our data implicates a direct role of B-cell intrinsic RANK signaling in MM pathogenesis. Further understanding of the molecular effects of RANK/RANKL in MM should help to optimize treatment combinations to reduce bone disease and target the malignant cells effectively, in order to provide more effective treatments to this deadly disease.

Competing endogenous RNA networks related to prognosis in chronic lymphocytic leukemia: comprehensive analyses and construction of a novel risk score model

BackgroundChronic lymphocytic leukemia (CLL) is a heterogeneous B-cell malignancy that lacks specific biomarkers and drug targets. Competing endogenous RNAs (ceRNAs) play vital roles in oncogenesis and tumor progression by sponging microRNAs (miRNAs). Nevertheless, the regulatory mechanisms of survival-related ceRNA networks in CLL remain to be uncovered.MethodsWe included 865 de novo CLL patients to investigate RNA expression profiles and Illumina sequencing was performed on four CLL patients, two CLL cell lines and six healthy donors in our center. According to univariate Cox regression, LASSO regression as well as multivariate Cox regression analyses, we established a novel risk score model in CLL patients. Immune signatures were compared between the low- and high-risk groups with CIBERSORT and ESTIMATE program. Afterwards, we analyzed the relationship between differentially expressed miRNAs (DEmiRNAs) and IGHV mutational status, p53 mutation status and del17p. Based on the survival analyses and differentially expressed RNAs with targeting relationships, the lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed. In addition, the circRNA circ_0002078/miR-185-3p/TCF7L1 axis was verified and their interrelations were delineated by dual-luciferase reporter gene assay.ResultsTotally, 57 differentially expressed mRNAs (DEmRNAs) and 335 DEmiRNAs were identified between CLL patient specimens and normal B cells. A novel risk score model consisting of HTN3, IL3RA and NCK1 was established and validated. The concordance indexes of the model were 0.825, 0.719 and 0.773 in the training, test and total sets, respectively. The high-risk group was related to del(13q14) as well as shorter overall survival (OS). Moreover, we identified DEmiRNAs that related to cytogenetic abnormality of CLL patients, which revealed that miR-324-3p was associated with IGHV mutation, p53 mutation and del17p. The survival-related lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed to further facilitate the development of potential predictive biomarkers. Besides, the expression of circ_0002078 and TCF7L1 were significantly elevated and miR-185-3p was obviously decreased in CLL patients. Circ_0002078 regulated TCF7L1 expression by competing with TCF7L1 for miR-185-3p.ConclusionsThe comprehensive analyses of RNA expression profiles provide pioneering insights into the molecular mechanisms of CLL. The novel risk score model and survival-related ceRNA networks promote the development of prognostic biomarkers and potential therapeutic vulnerabilities for CLL.

P1269: VIP152 IS A NOVEL CDK9 INHIBITOR WITH IMPROVED SELECTIVITY, TARGET MODULATION, AND CARDIAC SAFETY IN PATIENTS WITH LYMPHOMA

Background: Cyclin dependent kinase 9 (CDK9) controls transcriptional elongation, its inhibition may be effective in controlling cancer. To date, no drugs specifically targeting CDK9 have been approved due to challenges with therapeutic window. VIP152 was designed to be a highly potent and selective CDK9 inhibitor with favorable pharmacokinetic (PK) and pharmacodynamic (PD) effects to improve efficacy in patients with lymphoma. Aims: - To demonstrate the pharmacodynamic effects and safety profile associated with a selective CDK9 inhibitor with a favorable pharmacokinetic profile. - To demonstrate the utility of a selective CDK9 inhibitor for treatment of hematologic patients with an unmet medical need such as high-grade B cell lymphoma (HGBL) and chronic lymphocytic leukemia who relapse or are refractory to ibrutinib and venetoclax (R/R CLL). Methods: VIP152, fadraciclib, alvocidib (flavopiridol), KB-0742, and AZD4573 are evaluated in a CDK9/Cyclin T assay in the presence of low and high ATP and in a kinome scan at 1 µM. Differential expression (DE) analysis from VIP152, atuveciclib and KB-0742 treatment of 2 lymphoma cell lines by RNA seq is compared to DE of samples from 7 VIP152 treated HGBL patients. VIP152 cytotoxicity is evaluated in CRISPR/Cas9 edited HG-3 CLL cell line with homozygous TP53 edits R175H or R248Q, and in samples from 8 R/R CLL patients who relapsed or were refractory to ibrutinib and venetoclax. Results: In the presence of low ATP, the IC50 for VIP152 is 4.5 nM where others have IC50s from 3.2-29.4 nM. With high ATP, VIP152, AZD4573 and alvocidib maintain potency in low nM range, while fadraciclib and KB-0742 have 760 nM-1.7 µM IC50s. The kinome scan shows VIP152, KB-0742 and fadraciclib have the least number of hits. Kd values show VIP152 is the most selective CDK9 inhibitor versus all other CDKs. In patients at the current clinical dose of 30 mg administered once weekly, the average VIP152 unbound plasma concentration over a 24h period is 18 nM. The VIP152 IC50 of 4.5 nM unbound is maintained for 15h indicating target coverage. VIP152 PK exposure is dose-linear and proportional with low intra/inter-subject variability. Analysis of triplicate ECG data from 58 solid and heme cancer patients demonstrates that VIP152 does not prolong QTc interval after single or multiple doses (5-30 mg). DE analysis of MYC+ DLBLC cell lines with VIP152, atuveciclib or KB-0742 1µM treatment indicate that 85%, 76% or 68% of DE genes are downregulated, respectively and only VIP152 and atuveciclib achieve significant DE of MYC. DE is reproducible in HGBL patients’ whole blood at Cycle 1 Day 1 and Day 15 (r=0.9627) and findings will be validated in the ongoing clinical trial. VIP152 treatment demonstrates significant cytotoxicity in both WT and TP53 mutant CLL lines. R/R CLL samples treated with VIP152 showed a significant reduction in viability in a concentration-dependent manner. Summary/Conclusion: VIP152 is a potent and selective CDK9 inhibitor currently in development with a favorable cardiac safety profile. PK/PD demonstrates unbound plasma concentrations exceed in vitro IC50. Selective transcriptional downregulation is observed with 30mg IV weekly. Samples from patients with DLBCL or CLL are sensitive to VIP152 and updated PK, PD and ctDNA dynamics from the ongoing phase 1 trials (NCT02635672, NCT04978779) will be presented.

Pirtobrutinib inhibits wild-type and mutant Bruton’s tyrosine kinase-mediated signaling in chronic lymphocytic leukemia

Pirtobrutinib (LOXO-305), a reversible inhibitor of Bruton’s tyrosine kinase (BTK), was designed as an alternative strategy to treat ibrutinib-resistant disease that develops due to C481 kinase domain mutations. The clinical activity of pirtobrutinib has been demonstrated in CLL, but the mechanism of action has not been investigated. We evaluated pirtobrutinib in 4 model systems: first, MEC-1, a CLL cell line overexpressing BTKWT, BTKC481S, or BTKC481R; second, murine models driven by MEC-1 overexpressing BTKWT or BTKC481S; third, in vitro incubations of primary CLL cells; and finally, CLL patients during pirtobrutinib therapy (NCT03740529, ClinicalTrials.gov). Pirtobrutinib inhibited BTK activation as well as downstream signaling in MEC-1 isogenic cells overexpressing BTKWT, BTKC481S, or BTKC481R. In mice, overall survival was short due to aggressive disease. Pirtobrutinib treatment for 2 weeks led to reduction of spleen and liver weight in BTKWT and BTKC481S cells, respectively. In vitro incubations of CLL cells harboring wild-type or mutant BTK had inhibition of the BCR pathway with either ibrutinib or pirtobrutinib treatment. Pirtobrutinib therapy resulted in inhibition of BTK phosphorylation and downstream signaling initially in all cases irrespective of their BTK profile, but these effects started to revert in cases with other BCR pathway mutations such as PLCG2 or PLEKHG5. Levels of CCL3 and CCL4 in plasma were marginally higher in patients with mutated BTK; however, there was a bimodal distribution. Both chemokines were decreased at early time points and mimicked BCR pathway protein changes. Collectively, these results demonstrate that pirtobrutinib is an effective BTK inhibitor for CLL harboring wild-type or mutant BTK as observed by changes in CCL3 and CCL4 biomarkers and suggest that alterations in BCR pathway signaling are the mechanism for its clinical effects. Long-term evaluation is needed for BTK gatekeeper residue variation along with pathologic kinase substitution or mutations in other proteins in the BCR pathway.

LINC00152 expression in normal and Chronic Lymphocytic Leukemia B cells.

Long non‐coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naïve B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll‐like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients.

Abstract 3102: Venetoclax resistance associates with high-level expression of ROR1 and cancer stemness in chronic lymphocytic leukemia

Abstract The BH3-mimetic venetoclax is highly cytotoxic for chronic lymphocytic leukemia (CLL) cells. Nonetheless, some patients (pts) fail to clear minimal residual disease (MRD) and develop progressive disease (PD) on venetoclax-based therapy (V-Rx). Acquisition of de novo BCL2 mutations that reduce venetoclax-binding to BCL2 has been observed in CLL cells of pts who develop PD on V-Rx (Blombery et al., 2019; Blombery et al., 2020; Tausch et al., 2019). However, such BCL2 mutations generally are either subclonal or not invariably detected in CLL cells of pts who develop resistance to venetoclax (Blombery et al., 2019; Guieze et al., 2019). Moreover, CLL cells of pts with PD on V-Rx appear even more resistant to the cytotoxic effects of venetoclax when cultured under conditions mimicking stroma (Blombery et al., 2019; Thijssen et al., 2015), arguing that factors other than BCL2 mutations account for venetoclax resistance. We find that high-level expression of ROR1 and Wnt5a-induced ROR1-signaling causes enrichment in cancer-stemness gene expression in CLL. We examined whether this was associated with resistance to V-Rx. We performed flow cytometry and transcriptome analyses on CLL cells of pts prior to V-Rx (pre-Rx) and on CLL cells of the same pts after they developed PD on V-Rx after a median of 2 years of therapy. At PD, we identified de novo BCL2 mutations that were subclonal in 66% of these cases. Pre-Rx CLL cells of all 6 pts expressed high levels of ROR1, which became even higher on the CLL cells of the same pts after they developed PD on V-Rx. We found that that transcriptomes of CLL cells of pts with PD had even greater enrichment in cancer-stemness gene expression than pre-Rx CLL cells of these same pts. To examine whether expression of ROR1 could influence cancer stemness and resistance to venetoclax, we transfected the CLL-cell line MEC1 with ROR1 to generate MEC1-ROR1 cells. We found that MEC1-ROR1 had greater enrichment in cancer-stemness gene expression than MEC1 cells and had increased resistance to venetoclax in vitro. Moreover, mutant forms of BCL2 identified in CLL cells of pts with PD on V-Rx, and reducing venetoclax-binding to BCL2, were significantly more effective in protecting MEC1-ROR1 cells than MEC1 cells from the cytotoxic effects of venetoclax in vitro. We find that treatment of primary CLL cells with Wnt5a to induce ROR1-signaling could enhance their resistance to the cytotoxic effects of venetoclax in vitro; such induced-resistance could be inhibited by an anti-ROR1 mAb (cirmtuzumab), which can block Wnt5a-induced ROR1-signaling. Collectively these studies demonstrate that Wnt5a-induced ROR1-signaling in CLL promotes cancer dedifferentiation/stemness and increases resistance to Ven. Strategies that inhibit ROR1-signaling with agents such as cirmtuzumab may enhance the cytotoxic activity of venetoclax and/or mitigate risk of developing resistance to venetoclax therapy. Citation Format: Emanuela M. Ghia, Laura Z. Rassenti, Michael Y. Choi, Miguel Quijada-Alamo, Elvin Chu, George F. Widhopf, II, Thomas J. Kipps. Venetoclax resistance associates with high-level expression of ROR1 and cancer stemness in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3102.

BET Inhibitors in Chronic Lymphocytic Leukemia: JQ1 Synergizes with Venetoclax in Promoting Apoptosis

▪Background: The bromodomain and extra terminal BET family proteins (BRD2, BRD3, BRD4 and the testis-specific BRDT) play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Specifically, BET inhibitors block the transcription of key oncogenes (BCL2, C-MYC and CDK6) through the displacement of BRDs and other epigenetic modifiers from chromatin. However, while BET inhibitors were consistently described as highly effective drugs in in vitro experiments, few reports and clinical trials have already demonstrated the rapid emergence of BET inhibitors resistance.Aim: We examined the efficacy of the BET inhibitor JQ1 in CLL samples, upon short and prolonged treatments, to assess its efficacy and the potential development of resistances.Methods: MEC-1/EHEB CLL cell lines (p53 wild-type EHEB and p53-mutated MEC-1 cell lines) and primary CD19 positive lymphocytes from CLL patients were subjected to short and prolonged treatment with the BET inhibitor JQ1. Responses were measured in terms of proliferation, cell-cycle and apoptosis.Results: Treatment of CLL cell lines with the BET inhibitor JQ1 was associated with block of proliferation, cell cycle arrest and apoptosis. Molecularly, JQ1 treatment was associated to c-Myc down-modulation. Notably, treatment with JQ1 caused also loss of viability of primary patient-purified CLL cells. However, starting from 48 hours of JQ1 treatment in CLL cell lines, we observed a compensatory increase of BRDs mRNA levels, which was associated to the development of resistant clones after longer treatments (30 days). These data demonstrate that resistance to BET inhibitors can rapidly emerge and suggest that a combination therapy with other drugs should be explored. Interestingly, we observed that JQ1 was also able to modulate BCL-2 expression in CLL. We therefore assessed the effect of a combination therapy based on JQ1 in association with the BCL-2 inhibitor, Venetoclax. Intriguingly, co-treatment with JQ1 and Venetoclax significantly increased apoptosis demonstrating a synergic action.Conclusions: Our data demonstrated for the first time that: i) CLL may be effectively targeted by BET inhibitors; ii) short term treatment with BET inhibitors rapidly results in the upregulation of additional BRD family members; iii) prolonged treatment is associated with the development of resistance; IV) BET proteins modulate BCL-2 and c-MYC expression also in CLL; V) the combination therapy based of JQ1 administration in association with Venetoclax is extremely effective in CLLDiscussion: Here, we show that JQ1 treatment results in growth inhibition and apoptosis of CLL cells by down-modulating the expression of the pro-survival genes, C-MYC and BCL-2. Furthermore, our data show that treatment with JQ1 is only effective for a short time while the co-treatment with Venetoclax exerts a synergistic lethality effect in CLL. These data suggest the efficacy of a combination therapy with JQ1 and Venetoclax in refractory TP53 mutated CLL patients. DisclosuresSaglio:Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Wnt5a Induces ROR1 to Bind and Activate Cortactin, Which Functions in Planar Cell Polarity and Migration of Chronic Lymphocytic Leukemia Cells

Studies have revealed a dynamic trafficking of chronic lymphocytic leukemia cells (CLL) between the blood and lymphoid compartments. Targeted therapies that impair chemokine-receptor signaling, such as ibrutinib, disrupt the capacity of leukemia cells to re-enter lymphoid compartments, precluding CLL cells from receiving growth/survival signals from the accessory cells within the lymphoid-tissue microenvironment, thereby causing attrition in the leukemia-cell population over time in a patient undergoing such targeted therapy. Studying the signaling pathways and intracellular proteins that influence/govern leukemia-cell migration may identify additional agents that can further disrupt CLL-cell trafficking and potentially enhance the therapeutic activity of drugs such as ibrutinib. Factoring in leukemia-cell migration may be the cytoskeletal protein cortactin (also called EMS1), which possesses a SH3 domain and a F-actin-binding domain, allowing it link cell-surface receptor-complexes to the actin cytoskeleton. Upon tyrosine phosphorylation, cortactin is activated, thereby facilitating RhoGTPase activation and polymerization and rearrangement of the actin cytoskeleton, especially the actin cortex around the cellular periphery, which is required for planar cell polarity, lamellipodia formation, invadopodia formation, cell migration, and endocytosis. We find cortactin abundantly is expressed in CLL B cells. Moreover, in freshly isolated CLL cells, we find that cortactin associates with ROR1, an evolutionarily conserved, oncoembryonic surface-protein expressed on many cancers, including CLL. Culture of CLL cells in serum-free medium results in the dissociation/inactivation of the ROR1-cortactin complex unless the leukemia cells are stimulated with the ligand for ROR1, namely Wnt5a, which, relative to the plasma of age-matched healthy adults, is present at high levels in the plasma of patients with CLL, including patients undergoing therapy with ibrutinib. Moreover, we find that Wnt5a induces ROR1 to complex with cortactin, which then undergoes tyrosine phosphorylation at Y421, recruit ARHGEF1, and activate RhoA, thereby enhancing/enabling leukemia-cell migration. These effects of Wnt5a on CLL cells could not be inhibited by ibrutinib, but could be blocked by cirmtuzumab, a humanized, high-affinity monoclonal antibody specific for ROR1 that is undergoing clinical evaluation in patients with CLL. We corroborated these findings using the CLL cell line MEC1, which also expresses cortactin, but does not express ROR1. MEC1 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-cortactin complex formation. We confirmed that cortactin complexes with ROR1 and undergoes activation in response to Wnt5a in MEC1-ROR1 cells. We generated truncated forms of ROR1 and found the cytoplasmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with cortactin upon stimulation with Wnt5a. We next introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, 841, or 850 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P==>A mutants, ROR1P(841)A had impaired capacity to recruit cortactin to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate cortactin, and was not able to enhance MEC1-ROR1-cell F-actin polymerization. Collectively, these studies reveal that cortactin plays an important role in Wnt5a-enhanced CLL cell migration/F-actin polymerization via a ROR1-dependent signaling pathway that cannot be inhibited by ibrutinib or other inhibitors of BTK. DisclosuresKipps:AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Oncternal: Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Evaluation of membrane bound IL-21 expanded natural killer cells for chronic lymphocytic leukemia therapy

Abstract Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia and remains incurable despite recent therapeutic advances. Natural killer (NK) cells have potent anti-cancer effects, but efforts to use NK cell therapy in CLL have been limited by poor NK cell growth and anti-leukemia activity. We sought to determine if stimulating NK cells ex vivo with K562-based feeder cells expressing membrane-bound IL-21 (mbIL-21) (Denman et al., 2012) could overcome the limitations of prior techniques to create an effective therapy for CLL. Our data demonstrate that healthy donor-derived mbIL-21 expanded NK cells have increased antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity (DC; without targeting antibody) against CLL cell lines. This technique can also overcome CLL-induced NK suppression – CLL patient-derived NK cells expanded equally and were as potent as normal donor-derived NKs, including against both allogeneic and autologous CLL tumor cells. CLL tumor cells are not susceptible to DC but are very vulnerable to ADCC with anti-CD20 antibodies (rituximab or obinutuzumab). The lack of direct cytotoxicity of tumor cells was correlated to low NK:CLL quantitative conjugate formation, suggesting a lack of tumor target recognition. Blocking antibodies to FasL, TNFα, and TRAIL show that only TRAIL contributes modestly to anti-CLL tumor cytotoxicity, while application of folimycin demonstrates that perforin/granzyme release is the major mechanism of cytotoxicity. In vivo, treatment with expanded NK cells plus obinutuzumab significantly prolongs survival in the OSU-CLL human xenograft model. Based on these data, we are planning a clinical trial of mbIL-21 expanded NK cells plus obinutuzumab in CLL patients.

Identification of a Novel Role for PD-1 Signaling in Promotion Tumor Proliferation in B-Cell Lymphoma

Identification of a Novel Role for PD-1 Signaling in Promotion Tumor Proliferation in B-Cell Lymphoma

Abstract 3749: MER receptor tyrosine kinase overexpression potentiates survival signal in chronic lymphocytic leukemia cells

Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult B-cell leukemia in the United States. It is predominantly characterized by the accumulation of CD19+/CD5+/CD23+ monoclonal B-cells in peripheral blood, bone marrow and lymphoid tissues. BCR signaling pathway has emerged as a key driver for the expansion of neoplastic B-cell clones and pathogenesis in several B-cell malignancies including CLL. Stimulation of BCR induces activation of a cascade of downstream signal mediators including Bruton's tyrosine kinase (BTK), PI3K/AKT and ERK1/2. While the use of BCR-targeted agents, ibrutinib (BTK-inhibitor) or idelalisib (PI3Kδ-inhibitor), has been shown to be effective in relapsed/refractory CLL patients, the responses are limited to partial remissions. Therefore, insights into drugs with unique mechanism(s) of action that are nontoxic to the bone marrow/immune system and feasible in the older patient population (average age: 65 - 70 years) are urgently needed. To achieve such a goal it is critical to identify other targets that are constitutively active and may crosstalk with the BCR signal or other receptor tyrosine kinases (RTKs). MER RTK of the TAM (Tyro3, AXL, MER) family offers such an alternative approach. Previously, we detected that CLL B-cells express highly active AXL that acts as a docking site of multiple kinases/lipase including LYN, SYK/ZAP70, PI3K and PLCγ2 that, when subject to specific inhibition induces apoptosis. Most recently, we found that CLL B-cells from 50 - 60% of previously untreated CLL patients (n=30) also overexpressed MER at variable levels while, normal B-cells from health subjects did not show any such expression. Interestingly, MER in CLL B-cells remains constitutively phosphorylated (active). In addition, we also detected overexpression of its ligand, growth arrest specific gene 6 (Gas6) in CLL B-cells as compared to normal B-cells. Of note, leukemic B-cells from the Eμ-TCL1 transgenic mice which recapitulate aggressive human CLL also showed overexpression of MER. To this end, we found that exogenous addition of recombinant Gas6 to primary CLL B-cell culture or MEC1 (a CLL cell line) cells activated MER in a time-dependent manner. Interestingly, we also detected co-activation of BTK of the BCR pathway, following MER activation in CLL cells while, in vitro stimulation of BCR did not activate MER. Together, these findings suggest that MER activation may regulate the BCR signal in CLL B-cells in order to potentiate the survival signal. In conclusion, we detected overexpression of constitutively activated MER in CLL B-cells from some CLL patients which may represent high-risk CLL clones for clinical progression. Importantly, activation of MER may positively regulate the BCR signal inducing increased survival and apoptotic resistance of the leukemic B-cells to BCR-targeted agents. Further studies are in progress to elucidate the impact of targeting MER in CLL B-cell survival and resistance to therapy. Citation Format: Mariana T. Mendez, Hasan Mahmud, Sara K. Vesely, Jennifer Holter-Chakrabarty, Asish K. Ghosh. MER receptor tyrosine kinase overexpression potentiates survival signal in chronic lymphocytic leukemia cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3749.

Abstract 1756: JBI-802, novel dual inhibitor of LSD1-HDAC6 for treatment of cancer

Abstract Introduction: Lysine Specific Demethylase 1 (LSD1) is over-expressed in many cancers and down-regulation of LSD1 has been shown to effectively treat cancers by inducing re-expression of aberrantly silenced genes. Studies have shown that LSD1 may contribute to acute myelogenous leukaemia pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML. In addition, LSD1 inhibition also leads to convertion of ‘cold' tumors to ‘hot' tumors by activating type 1 interferon response. Similarly, HDAC6 inhibition enhances immune response by over-coming immune suppression. Further, combined inhibition of LSD1 and HDAC has been shown to be more efficacious in inhibiting multiple cancers. Here, we show that JBI-802, a dual inhibitor targeting both LSD1 and HDAC6/8 shows stronger efficacy in several cancers, including AML, lymphoma and others and well tolerated at efficacious doses. Methods: Computational chemistry approaches were used to design LSD1 specific and LSD1-HDAC dual inhibitors. To assess in vitro LSD1 potency, TR-FRET assay was used. For assessing in vitro HDAC activity fluorescence based HDAC6 activity assay was performed. Western blotting and quantitative PCR were used to assess biomarkers of LSD1 and HDAC inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Xenograft and syngeneic models were used to assess in vivo efficacy. Results: Several compounds from this series show strong in vitro potency against LSD1 with excellent selectivity against MAOs. JBI-802 our lead dual molecule, showed an IC50 of 0.05 μM against LSD1 and isoform selective HDAC6/8 activity (IC50 of 0.011 and 0.098 µM on HDAC6 and HDAC8, respectively), with about 100 fold selectivity against other HDAC isoforms. When assessed in a panel of 25 cell lines, JBI-802 showed strong anti-proliferative activity on select AML, CLL, SCLC, sarcoma and multiple myeloma cell lines. In cell based and in vivo target engagement studies there was significant dose-dependent increase in CD11b, CD86 and GFI1b and tubulin acetylation levels. JBI-802 had a much stronger effect in inhibiting the growth of HEL92.1.7 erythroleukemia xenograft by oral administration when compared to ACY-1215, a HDAC6 selective inhibitor or ORY-1001 an LSD1 selective inhibitor. ED50 of JBI-802 in this model was ~6.25 mg/kg BID. Further, dose-dependent modulation of above mentioned biomarkers and inhibition of c-Myc could be observed in tumors. Stronger tumor growth inhibition was also observed in other haematological cancers models such as CMK-1 (leukemia) and Z-138 (lymphoma). In addition, JBI-802 showed comparable single agent activity to anti-PD-1/PD-L1 mAbs in a syngeneic murine colon cancer model CT26 and resulted in stronger tumor growth inhibition when combined with these antibodies. 14-day dose ranging finding toxicology studies clearly show that the molecule was well tolerated up to 300 mg/kg. Conclusion: The data obtained to date demonstrate that dual LSD1-HDAC6/8 inhibitors could serve as novel, effective therapeutic agents for treatment of select subset of AML and other cancers. IND-enabling studies are in progress with this inhibitor to be developed as a clinical candidate Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Sreekala Nair, Basavaprabhu B, Reshma Dhkar, Santosh Viswakarma, Amir Siddiqui, Mohd Zainuddin, Rudresh G, Prashanthi Daram, Sunil Mohire, Krishnakumar V. JBI-802, novel dual inhibitor of LSD1-HDAC6 for treatment of cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1756.

Abstract 3305: Comparative analysis of RNA versus DNA as input material for IGH repertoire sequencing based detection of rare clonal B cells at a frequency of 10E-6

Abstract Background: Next generation sequencing of rearranged IGH chains has emerged as a reproducible and highly sensitive approach for detecting rare B cell clones, e.g. malignant B cell, in peripheral blood. Historically, efforts to evaluate the frequency of rare B cells by IGH chain sequencing have utilized DNA input given potential challenges in accurately quantifying template copy number from RNA data owing to B cell subtype specific variation in the expression of the B cell receptor. Hypothetically, however, RNA input based monitoring could be advantageous both owing to reduced input requirements and superior ability to detect B cell malignancies of plasmablast and plasma cell origin, where the BCR is robustly expressed. Here we compared the ability of RNA and DNA based IGH chain sequencing to detect two Burkitt's Lymphoma B cell lines (CA46 and GA10) at a frequency of 10E-6 from peripheral blood. We discuss advantages of either approach for detection of rare B cell clones. Methods: IGH chain libraries were prepared using the Oncomine IGH-SR assay (framework 3 and joining gene multiplex primers) from gDNA or total RNA extracted from peripheral blood and spiked with Burkitt's lymphoma or CLL cell line gDNA or total RNA to a frequency of 10E-6 by mass ratio. Libraries were sequenced via the Gene Studio S5 followed by Ion Reporter analysis to identify clonotypes and evaluate B cell clone frequencies across samples. Automated downsampling analysis was used to confirm libraries were sequenced to saturation. Library preparation and analysis was performed in replicate to quantify sensitivity of detection. Results: For each cell line, we prepared and sequenced (1) 30 libraries derived from amplification of 2ug gDNA spiked with 2pg cell line gDNA and (2) 10 libraries derived from amplification of 100ng RNA spiked with .1pg cell line total RNA. The Burkitt's lymphoma cell lines were detected in 10/30 and 8/30 gDNA libraries respectively, for CA46 and GA-10) consistent with the historical performance of orthologous DNA-based sequencing approaches. For RNA libraries, the Burkitt's lymphoma cell lines were detected in each library (10/10 and 10/10, respectively). Conclusions: Here we demonstrate detection of B cell lines at a frequency down to 10E-6 from DNA and RNA input. Importantly, we find that RNA based IGH sequencing may significantly reduce input requirements for rare clone detection, potentially enabling routine detection of clones down to 10E-6 frequency from a single library. Citation Format: Michelle Toro, Loni Pickle, Jayde Chang, Timothy Looney, Geoffrey Lowman, Mark Andersen, Fiona Hyland. Comparative analysis of RNA versus DNA as input material for IGH repertoire sequencing based detection of rare clonal B cells at a frequency of 10E-6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3305.

Exosomes derived from chronic lymphocytic leukaemia cells transfer miR-146a to induce the transition of mesenchymal stromal cells into cancer-associated fibroblasts.

Chronic lymphocytic leukaemia (CLL) is the most prevalent leukaemia and remains incurable. Mesenchymal stem cells (MSCs) can promote tumour progression by differentiating into cancer-associated fibroblasts (CAFs). However, the mechanisms by which tumour cells induce the transition of MSCs to CAFs are still largely undefined. Exosomes can regulate recipient cellular function by mediating intracellular communication. This study aimed to investigate whether CLL cells regulate the transition of bone marrow-derived MSCs (BM-MSCs) to CAFs via exosomal miR-146a delivery. The exosomes were isolated from CLL cell line MEC-1 (CLL-Exo) and then co-cultured with BM-MSCs. The expression of α-smooth muscle actin (α-SMA) and fibroblast-activated protein (FAP) were determined by immunofluorescence, quantitative real-time polymerase chain reaction and western blot. A luciferase reporter assay was performed to verify whether ubiquitin-specific peptidase 16 (USP16) was a target of miR-146a. CLL-Exo treatment up-regulated miR-146a and down-regulated expression of CAF markers (α-SMA and FAP) and USP16. The inducing effect of CLL-Exo on CAF marker expression was compromised when miR-146a expression was inhibited in CLL-Exo. USP16 was confirmed as a direct target of miR-146a and USP16 overexpression in BM-MSCs abrogated the CLL-Exo-mediated up-regulation of CAF markers. Collectively, CLL-Exo delivered miR-146a into BM-MSCs where miR-146a mediated transition of BM-MSCs into CAFs by targeting USP16.

Lineage Tracing in Chronic Lymphocytic Leukemia Reveals Clones with Stable Gene Expression States That Differentially Respond to Therapy

Cancer's ability to evolve and adapt is a major challenge to therapeutic success. Fueling this evolution is vast tumor heterogeneity, with constituent clones varying in their genetics, epigenetics and response to therapy. As a field, however, we have yet to couple the genetic alterations present within individual clones to their transcriptional or functional outputs. Here, we applied a novel adaptation of clone tracing that integrates DNA barcoding with single-cell RNA sequencing (scRNA-seq) to HG3, a CLL cell line harboring del(13q) and no other known cancer drivers, to model in vitro responses to front-line chemotherapy with fludarabine and cyclophosphamide at clone-level resolution. To generate a high-diversity barcode library compatible with scRNA-seq, a random pool of 20 base pair DNA barcodes was introduced into the 3'UTR of a reporter gene in a lentiviral expression vector. This viral barcode library was transduced into HG3 cells (at MOI 0.1 to minimize multiple barcode-tagging of cells) and 1.2x106 barcoded cells were sorted and expanded to establish the parental barcoded HG3 population. We subsequently treated this barcoded population with an LD95 combined dose of fludarabine and mafosfamide (the in vitro analog of cyclophosphamide) in 8 parallel experiments. Cell barcodes were sequenced prior to treatment (TP1) and following outgrowth from treatment (TP2) for analysis of clonal composition. 10,000 cells each from TP1 and from 2 of 8 parallel replicates at TP2 were processed for scRNA-seq. We observed a massive decrease in viability across all 8 replicates, with regrowth occurring at 20 days post-treatment. Barcode analysis revealed a marked decrease in clonal diversity from TP1 to TP2 (11,827 to 2,622 ± 380, n=8; or ~78%), and clones that survived treatment did so consistently such that 94% of surviving cells in each replicate had a clonal identity that was present in all 8 replicates. Analysis of clonally-resolved transcriptional profiles revealed that clones consistently fell into one of two stable gene expression states (clusters) prior to treatment, with nominal intermixing between populations. Treatment predominantly selected for clones comprising the smaller of these two clusters (TP1-'high tolerance'), with only a minimal number of resistant clones originating from the larger cluster at TP1 (TP1-'low tolerance'). Pathway and gene set enrichment analysis of these two TP1 clusters demonstrated that TP1-high tolerance had a stark upregulation of common CLL signaling pathways (i.e. WNT and CXCR4, an inflammatory/migratory phenotype) and a reliance on chromatin modification pathways. TP1-low tolerance, on the other hand, exhibited upregulated type 2 antigen presentation and prostaglandin biosynthesis/metabolism which has a known role in driving inflammation and migration in adjacent cells (Wang et al, BMJ 2006). These gene expression states remained stable after treatment, but with the added upregulation of well-described mechanisms of resistance to cyclophosphamide, with TP1-high tolerance exhibiting upregulation of GSTP1, a glutathione S-transferase that is a main mediator of cyclophosphamide metabolism, and TP1-low tolerance exhibiting upregulation of members of the ALDH family thought to ameliorate toxicity from chemotherapy-induced ROS (Andersson et al, Acta Oncologica 1995). Through this work, we resolved the underlying clonal composition of a CLL cell line and observed that the constituent clones exhibit stable and discrete gene expression states that differentially respond to chemotherapy. We noted two different axes of resistance - a more successful avenue that relies on WNT and CXCR4 signaling as well as cyclophosphamide clearance for resistance, and a less-successful avenue that involves clearance of reactive oxygen species for survival. The intersection of these two critical CLL pathways in in vitro resistance to first-line CLL therapy is of particular interest given the FAT1 (WNT regulator) mutations and CXCR4 upregulation frequently seen in chemo-refractory CLL (Messina et al, Blood 2014; Burger et al, Blood 2006), and future efforts will assess the stability and interplay of these two pathways in patient samples collected upon relapse to fludarabine and cyclophosphamide. Further, our approach provides a template for the high-resolution study of tens of thousands of clones and their respective phenotypes in a mixed leukemic population. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Pharmacyclics: Research Funding; Neon Therapeutics: Other: Member, Advisory Board.