Chronic Lymphocytic Leukemia Cell Viability Research Articles

Anti-tumor activity of selinexor in combination with antineoplastic agents in chronic lymphocytic leukemia

Despite recent relevant therapeutic progresses, chronic lymphocytic leukemia (CLL) remains an incurable disease. Selinexor, an oral inhibitor of the nuclear export protein XPO1, is active as single agent in different hematologic malignancies, including CLL. The purpose of this study was to evaluate the anti-tumor effects of selinexor, used in combination with chemotherapy drugs (i.e. fludarabine and bendamustine) or with the PI3Kδ inhibitor idelalisib in CLL. Our results showed a significant decrease in CLL cell viability after treatment with selinexor-containing drug combinations compared to each single compound, with demonstration of synergistic cytotoxic effects. Interestingly, this drug synergism was exerted also in the presence of the protective effect of stromal cells. From the molecular standpoint, the synergistic cytotoxic activity of selinexor plus idelalisib was associated with increased regulatory effects of this drug combination on the tumor suppressors FOXO3A and IkBα compared to each single compound. Finally, selinexor was also effective in potentiating the in vivo anti-tumor effects of the PI3Kδ inhibitor in mice treated with the drug combination compared to single agents. Our data provide preclinical evidence of the synergism and potential efficacy of a combination treatment targeting XPO1 and PI3Kδ in CLL.

Therapeutic Targeting Potential of Novel Silver Nanoparticles Coated with Anti-CD20 Antibody against Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable disorder associated with alterations in several pathways essential for survival and proliferation. Despite the advances made in CLL therapy with the new target agents, in some cases, relapses and resistance could occur, making the discovery of new alternatives to manage CLL refractoriness necessary. To provide new therapeutic strategies for CLL, we investigated the anti-leukemic activity of silver nanoparticles (AgNPs), whose impact on CLL cells has been poorly explored. We studied the action mechanisms of AgNPs in vitro through flow cytometry and molecular analyses. To improve the bioavailability of AgNPs, we generated AgNPs coated with the anti-CD20 antibody Rituximab (AgNPs@Rituximab) and carried out imaging-based approaches and in vivo experiments to evaluate specificity, drug uptake, and efficacy. AgNPs reduced the viability of primary CLL cells and the HG-3 cell line by inducing an intrinsic apoptotic pathway characterized by Bax/Bcl-2 imbalance, caspase activation, and PARP degradation. Early apoptotic events triggered by AgNPs included enhanced Ca2+ influx and ROS overproduction. AgNPs synergistically potentiated the cytotoxicity of Venetoclax, Ibrutinib, and Bepridil. In vitro, the AgNPs@Rituximab conjugates were rapidly internalized within CLL cells and strongly prolonged the survival of CLL xenograft models compared to each unconjugated single agent. AgNPs showed strong anti-leukemic activity in CLL, with the potential for clinical translation in combination with agents used in CLL. The increased specificity of AgNPs@Rituximab toward CLL cells could be relevant for overcoming in vivo AgNPs' non-specific distribution and increasing their efficacy.

Functional Anti-Apoptotic Protein Dependence and Its Relation to Genomic Prognostic Markers and Response to Treatment in Chronic Lymphocytic Leukemia

Introduction Due to its heterogenous biology, chronic lymphocytic leukemia (CLL) is a well-suited disease for identifying new relationships between genomic and functional biomarkers for drug response. Here, we combined a multi-omics approach and ex vivo drug-treatment data with functional characterization of apoptotic sensitivity to correlate genetic features and drug sensitivity with specific anti-apoptotic signaling patterns measured by BH3 profiling in primary CLL samples. Methods BH3 profiling is a functional assay to measure the proximity of cells to undergo apoptosis via overall priming as well as to identify specific anti-apoptotic proteins the cells are dependent on for survival. BH3 peptides and mimetic such as BIM and PUMA were used to identify overall priming or proximity to apoptosis, BAD and ABT199 were used to identify BCL-2 dependence, and FS1, MS1 and HRK were used to identify BFL-1, MCL-1 and BCL-xL dependences respectively (Ryan et al, Biol Chem, 2016). Results We analyzed primary CLL samples from 73 patients and using BH3 profiling identified the specific associations between anti-apoptotic protein dependence and genetic features (such as IGHV status, trisomy-12, del(17p) and TP53 mutation. Our univariate models demonstrated that BCL-2 dependence, as measured by ABT199 drug (p=0.001) and BAD (p=0.004) BH3 peptide, are highly correlated with mutated IGHV status, a favorable prognostic marker in CLL (Figure 1A). Using ex vivo treatment with multiple classes of small molecule inhibitors, we further identified several specific drug response or sensitivity patterns correlating with anti-apoptotic protein dependence as well as CLL prognostic factors (Figure 1B). In particular, our multivariate models (LASSO regularized) demonstrated that viability following ex vivo treatment with the MDM-2 inhibitor Nutlin-3 was positively correlated with TP53 mutation/deletion, but inversely correlated with BFL-1 (FS1 peptide) and BCL-xL (HRK peptide) dependence as well as trisomy-12 (Figure 1B), though the inverse correlation between Nutlin-3 and BFL-1 or BCL-xL dependence is relatively weak. CLL cell viability following ex vivo treatment with the dual BCL-2/BCL-xL inhibitor navitoclax was negatively correlated with BCL-2 (BAD peptide) and BCL-xL (HRK peptide) dependence, though primarily BCL-2 dependence, suggesting that sensitivity to navitoclax is highly dependent on BCL-2 (Figure 1B). At the transcriptomic level, CLL cell dependence on BCL-2 positively correlated with NOXA expression, NFκB signaling, and reduced intracellular oxidative level, various pathways that could potentially maintain BCL-2 dependence. Finally, we utilized BH3 profiling to retrospectively predict in vivo drug response, with high BCL-2 dependence (p=0.016) correlating with greater absolute lymphocyte count decrease. Conclusion Functional characterization of apoptotic signaling in CLL demonstrated that patients with mutated IGHV are more dependent on BCL-2 for survival, and that BH3 profiling could retrospectively predict in vivo sensitivity to BCL-2 inhibition. This functional precision medicine approach may help to stratify patients with CLL beyond genetic markers and should be further explored for correlation with clinical outcomes in prospective clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

NOTCH1 Signaling Is Dysregulated in CLL By the Deubiquitinase USP28 That Is Recurrently Affected By Del(11q) and Can be Therapeutically Targeted

Active NOTCH1 signaling is present in approximately half of chronic lymphocytic leukemia (CLL) patients and was shown to be a marker for disease outcome. The NOTCH1 signaling pathway can be aberrantly activated through mutations in the NOTCH1 gene itself or co-factors such as FBXW7, resulting in protein stabilization of cleaved NOTCH1 (NICD). However, 50% of CLL cases with NOTCH1 activation do not harbor alterations in these genes, suggesting additional mechanisms for NOTCH1 hyperactivation. Since NICD can be stabilized due to defects in the ubiquitination/degradation machinery, we aimed to elucidate the role of the on chromosome 11q23 located deubiquitinase USP28 in the regulation of NOTCH1 activity and as a novel therapeutic target for NOTCH1-dependent CLL patients. First, we evaluated USP28 expression levels in a previously published cohort of 285 untreated CLL patients characterized via SNP array analysis and gene expression profiling (Edelmann et al, Blood 2012; Bloehdorn et al, Nat Commun 2021). The presence of monoallelic USP28 deletion was identified in 90% of CLL patients harboring a heterozygous deletion of chromosome 11q (del(11q); n=96), resulting in a significant decrease of USP28 RNA expression in comparison to non-del(11q) cases (n=199, P<0.0001). In non-del(11q) cases, USP28 expression levels were variable and assessment of protein levels of USP28 and its target proteins by western blotting in primary CLL cells (n=8) revealed a correlation between high protein levels of USP28 and remarkably high levels of NICD and other USP28 targets such as c-MYC or c-JUN. In addition, interaction of USP28 with NICD was shown by immunoprecipitation assays. In parallel, we implemented the CRISPR/Cas9 system to generate HG3 CLL-derived cell lines harboring USP28 biallelic deletion (HG3-USP28KO, n=5), monoallelic 11q deletion (HG3-del(11q), 17 Mb size including both ATM and USP28 genes, n=3), biallelic ATM truncation (HG3-ATMKO, n=3) or the combination of both events (HG3-del(11q) ATMKO, n=3). Overexpression of USP28 in HG3WT and HG3-USP28KO cells increased NOTCH1 activity measured by luciferase reporter assays (P<0.05, P<0.001, respectively). In contrast, in HG3-del(11q) cells decreased USP28 RNA and protein expression resulted in reduced NOTCH1 activity compared to HG3WT cells (P<0.05). Notably, HG3-del(11q) ATMKO cell lines showed even lower levels of NOTCH1 activity (P<0.01). Given that ATM phosphorylates USP28 (Zhang et al, Cell 2006), we hypothesized that in CLL cells ATM regulates USP28 activity and thereby also downstream NOTCH1 signaling. Indeed, NOTCH1 activity was reduced in HG3-ATMKO cells (P<0.01). However, USP28 overexpression in HG3-ATMKO cells did not lead to increased NOTCH1 signaling activity as observed in HG3WT cells. Moreover, pharmacological ATM inhibition (5 µM of KU-55933) also resulted in reduced NOTCH1 activity in HG3WT cells (P<0.0001) and reduced NICD levels in primary CLL cells (n=12; P<0.05). We finally explored the impact of the small molecule USP28 inhibitor AZ1 (alone or in combination with ibrutinib or venetoclax) on NICD protein levels and viability of primary CLL cells (n=15). AZ1 treatment (10 µM) resulted in decreased NICD levels, independent of mutations within NOTCH1 or FBXW7 (P<0.01). ATP quantification and 7-AAD staining revealed that increasing doses of AZ1 significantly reduced viability of NOTCH1/FBXW7-MUT primary cells (n=5) as well as of NOTCH1/FBXW7-WT cells (n=6) expressing high protein levels of USP28 and NICD (P<0.01; P<0.05, respectively). In contrast, AZ1 treatment did not affect viability of CLL patient cells expressing low NICD levels (n=4; P=0.56) Furthermore, AZ1 combined with venetoclax potentiated the reduction of cell viability especially in NOTCH1/FBXW7-MUT or patient cells expressing high USP28 and NICD levels (P<0.001; P<0.05). We propose that high levels of the deubiquitinase USP28 are responsible for hyperactivation of NOTCH1 signaling in CLL. This hypothesis is further confirmed in del(11q) CLL cells, where USP28 heterozygous deletion resulted in low NOTCH1 activity. In addition, our data reveal that USP28 activity is regulated by ATM, uncovering a novel mechanism of NOTCH1 modulation via the ATM-USP28-NOTCH1 axis (Figure 1). Finally, we demonstrate that USP28 inhibition by AZ1 suppresses USP28-mediated NOTCH1 activation and could represent a novel therapeutic strategy in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combined Treatment with Ibrutinib and Anti-CD38 Monoclonal Antibody Daratumumab in Relapsed/Refractory Chronic Lymphocytic Leukemia with TP53 Aberrations: Results of the Filo Phase II Study IDA53

Background: Despite significant improvement with BTK inhibitors, long-term disease control of relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) with TP53 abnormalities remains challenging. CD38 expression is an independent adverse prognostic factor in CLL. This has been related to the involvement of CD38 in CLL cells migration, proliferation, tissue invasion and BCR signaling (Vaisiti 2015; Matas-Cespedes 2016). Daratumumab is the first in class anti-CD38 monoclonal antibody approved in multiple myeloma in both relapse and frontline settings. Preclinical studies have shown daratumumab to induce significant effect on viability and migration of patient-derived CLL cells (Matas-Céspedes et al, 2016). Moreover, the combination of daratumumab and ibrutinib demonstrated synergistic effect on both in vitro CLL cells killing and in vivo tumor burden reduction and survival prolongation in a CLL xenograft model (Manna et al, 2019). We therefore hypothesized that daratumumab would improve ibrutinib efficacy in R/R CLL patients with TP53 aberrations. Methods: This phase 2 trial enrolled patients with R/R CLL and TP53 abnormalities regardless of cell surface CD38 expression. Treatment consisted of ibrutinib (420 mg/d) alone for one month then combined to intravenous daratumumab (16 mg/kg/d) administered weekly for 8 weeks, every other week for the subsequent 4 months then monthly until intolerance or disease progression. The primary endpoint was the complete response (CR) rate according to iwCLL criteria after 12 months (M12) of combined therapy. We estimated a 20% CR rate at M12 to be the target threshold for treatment efficacy and 7% the minimum acceptable. We planned to recruit 44 patients. Results: Between December 2018 and May 2022, 29 patients have been enrolled. The study prematurely closed due to drop in patient accrual and failure to achieve the primary endpoint. Median age was 69 years [45-86] and sex ratio M/F was 18/11. Median number of previous lines was 1 [1-4]. Four patients (14%) had bulky adenopathy. Median lymphocytosis was 55 G/L (4.5-385). Median hemoglobin level and platelet count were 13.2 g/dL (8.1-17.3) and 127 G/L (37-188) respectively. All patients had TP53 disruption with 17p deletion in 16 cases (55%), TP53 mutations in 26 (93%), both in 14 (48%). Five had 11q deletion (18.5%). IGHV was unmutated in 16 patients (62%) and 2 additional patients had subset#2 stereotyped BCR. Eighteen patients had complex karyotype (72%) of whom 9 (36%) had very complex karyotype. Elevated b2microglobulin was present in 19 patients (73%). CD38 expression ranged from 0 to 87%. At data cutoff (18 JUL 2022), 21 patients have reached M12 and 15 are still on treatment. Fourteen patients went off study of whom 6 before M12. Reasons of discontinuation were progressive disease (PD) in 4 patients (at month 4, 13, 19 and 25) of whom 2 Richter transformation (RT), related adverse event (AE) in 3 (grade 3 infusion related reaction (IRR), recurrent cutaneous carcinoma, atrial fibrillation), COVID-19 in 3, pandemic context in 1, death of unknown cause in 1, and consent withdrawal in 2 patients. Five patients died (1 RT, 3 COVID-19, 1 unknown cause). AEs and serious AEs (SAE) were reported in 29 and 19 patients, respectively. Grade ≥3 AEs were hematological (n=9), infections (n=7), metabolism disorder (n=5), IRR, renal deficiency, myalgia, urologic disorder, death of unknown cause (all n=1). Grade 1-2 AEs reported in ≥ 10% of patients included gastrointestinal disorder (n=16), metabolism disorder (n=10), cardiovascular disorder (n=7), cutaneous neoplasms (n=6), ENT infections (n=5), COVID 19 (n=5), bleedings (n=4) and skin disorders (n=3). No tumor lysis syndrome occurred. At M12, one patient achieved CR, 20 patients achieved PR. Overall response and CR rate were 78% and 3.7% in intend-to-treat calculation, and 95% and 4.5% in per protocol calculation, respectively. With a median FU of 20.2 months, median progression free and overall survival were 27.1 [95%CI 23.4-NR] and 33.5 [95%CI 27.1-NR] months, respectively. Although not significant, there was a trend toward shorter PFS in CD38+ patients that daratumumab did not reverse. Conclusions: Daratumumab was safely combined with ibrutinib without unexpected toxicity in CLL. However, this combination did not provide any clear improvement of complete response rate in this very high-risk CLL population, as compared with ibrutinib alone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3β downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3β silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3β mutant reduces them. Additionally, the GSK3β inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3β in CLL cells. SB216763 reduces GSK3β/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3β is less active. We then modulated the activity of two upstream regulators of GSK3β and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3β and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3β, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3β activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3β a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential.

Notch2 Increases the Resistance to Venetoclax-Induced Apoptosis in Chronic Lymphocytic Leukemia B Cells by Inducing Mcl-1.

Chronic lymphocytic leukemia (CLL) has experienced a clinical revolution—thanks to the discovery of crucial pathogenic mechanisms. CLL is still an incurable disease due to intrinsic or acquired resistance of the leukemic clone. Venetoclax is a Bcl-2 inhibitor with a marked activity in CLL, but emerging patterns of resistance are being described. We hypothesize that intrinsic features of CLL cells may contribute to drive mechanisms of resistance to venetoclax. We analyzed the expression of Interferon Regulatory Factor 4 (IRF4), Notch2, and Mcl-1 in a cohort of CLL patients. We evaluated CLL cell viability after genetic and pharmaceutical modulation of Notch2 expression in patients harboring trisomy 12. We tested venetoclax in trisomy 12 CLL cells either silenced or not for Notch2 expression or in combination with an inhibitor of Mcl-1, AMG-176. Trisomy 12 CLL cells were characterized by low expression of IRF4 associated with high levels of Notch2 and Mcl-1. Notch2 and Mcl-1 expression determined protection of CLL cells from spontaneous and drug-induced apoptosis. Considering the involvement of Mcl-1 in venetoclax resistance, our data demonstrated a contribution of high levels of Notch2 and Mcl-1 in a reduced response to venetoclax in CLL cells carrying trisomy 12. Furthermore, reduction of Mcl-1 expression by silencing Notch2 or by treatment with AMG-176 was able to restore the response of CLL cells to venetoclax. The expression of Notch2 identifies a subset of CLL patients, mainly harboring trisomy 12, characterized by high levels of Mcl-1. This biological mechanism may compromise an effective response to venetoclax.

IL-10 Rescues CLL Survival through Repolarization of Inflammatory Nurse-like Cells.

Simple SummaryIn in vitro co-cultures of CLL cells and nurse-like cells (NLC), protection against apoptosis is only provided by M2-like NLC, and not M1-like NLC. In this study, we propose that fine-tuning of NLC polarization (and therefore survival of leukemic cells) is dictated by a balance between IL-10 and TNF.Tumor-associated macrophages (TAMs) in chronic lymphocytic leukemia (CLL) are also called nurse-like cells (NLC), and confer survival signals through the release of soluble factors and cellular contacts. While in most patient samples the presence of NLC in co-cultures guarantees high viability of leukemic cells in vitro, in some cases this protective effect is absent. These macrophages are characterized by an “M1-like phenotype”. We show here that their reprogramming towards an M2-like phenotype (tumor-supportive) with IL-10 leads to an increase in leukemic cell survival. Inflammatory cytokines, such as TNF, are also able to depolarize M2-type protective NLC (decreasing CLL cell viability), an effect which is countered by IL-10 or blocking antibodies. Interestingly, both IL-10 and TNF are implied in the pathophysiology of CLL and their elevated level is associated with bad prognosis. We propose that the molecular balance between these two cytokines in CLL niches plays an important role in the maintenance of the protective phenotype of NLCs, and therefore in the survival of CLL cells.

PI3K-Delta Inhibitors Induce Primary Monocyte Cytotoxicity but Do Not Alter Monocyte Differentiation

It is well appreciated that an altered tumor microenvironment is a hallmark of cancer, including in chronic lymphocytic leukemia (CLL) where monocyte-derived cells known as “nurse-like cells” (NLC) in the lymph node microenvironment support CLL cell viability, survival, and resistance to drug-induced apoptosis. CLL directed therapy may include conventional chemotherapy, immunotherapy, targeted kinase inhibitors, and/or Bcl2 inhibitors. Of these options, kinase inhibitors such as PI3K-delta inhibitors are an effective therapy that have been found to disrupt NLC-CLL cell interaction in vitro and induce initial lymphocytosis thought to be related to this disrupted cellular interaction. We previously demonstrated that the PI3K-delta inhibitors idelalisib and umbralisib (TGR-1202) induce cytotoxicity, apoptosis, and inhibition of pAKT in primary CLL cells. We now investigate the effect of idelalisib and umbralisib on monocytes by evaluating cytotoxicity and cytokine-induced differentiation. We isolated monocytes from blood obtained from normal donors using negative selection (RosetteSep monocyte). For cytotoxicity experiments, primary purified monocytes were incubated ± M-CSF (10 ng/mL) ± PI3K-delta inhibitor (at 1.25 to 20 μM) for three days, and cell viability was measured with the MTS reagent after three hours. For differentiation experiments, primary purified monocytes were incubated first with M-CSF (10 ng/mL) for three days, then washed and incubated ± IL-10 (20 ng/mL) ± PI3K-delta inhibitor (10 μM) for three days. To judge monocyte differentiation, we used flow cytometry to measure expression of CD14, CD206, CD163, CD124, CD80, and CD86. We also visually assessed monocyte appearance using phase microscopy with these conditions. Both idelalisib and umbralisib induced concentration dependent cytotoxicity (N = 3), but cytotoxicity was higher with idelalisib (62% cytotoxicity at 1.25 μM) compared to umbralisib (24% cytotoxicity at 1.25 μM). The addition of M-CSF reduced PI3K-delta inhibitor-induced cytotoxicity of primary monocytes. For example, 1.25 μM idelalisib induced 62% cytotoxicity without M-CSF, compared with 5% cytotoxicity with M-CSF. Staged cytokine incubation of monocytes with M-CSF and IL-10 induced the expression of M2 differentiation markers (CD163 and CD124) and reduced expression of the M1 differentiation marker (CD86). Addition of idelalisib or umbralisib did not significantly affect monocyte surface marker expression compared to no drug (N = 4). Even though idelalisib and umbralisib were cytotoxic for monocytes, these drugs did not affect morphologic changes induced by IL-10 (marked spreading and shape change). Thus, PI3K-delta inhibitors do not appear to affect cytokine-induced monocyte differentiation, but they do induce monocyte cytotoxicity (idelalisib > umbralisib). The addition of M-CSF, which contributes to M2 differentiation, blocks these effects. Together, our results suggest that depleting monocyte or NLC number may contribute to the anti-CLL therapeutic effects of PI3K-delta inhibitors. In addition, a higher level of monocyte cytotoxicity with idelalisib compared to umbralisib could potentially contribute to the higher infectious side effect profile with idelalisib seen in clinical use, as monocyte derived cells are a key component of the innate immune system. The anti-monocyte effect of PI3K-delta inhibitors could provide rationale for using these agents in other malignancies in which monocyte-derived cells are an integral part of the tumor microenvironment. DisclosuresFriedman:TG Therapeutics: Research Funding. Maryanski:TG Therapeutics, Inc.: Employment, Equity Ownership. Miskin:TG Therapeutics, Inc.: Employment, Equity Ownership.

CDK7 Inhibitor THZ1 Inhibits Glycolytic Metabolism By Suppressing HIF1α Activity in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL), the most common type of leukemia in Western adults, is characterized by the progressive accumulation of CD19+ malignant B cells in blood, secondary lymphoid tissues and bone marrow. Currently, CLL remains incurable in spite of the explosive development of novel therapeutics in the past decade. Cyclin-dependent kinases (CDKs) have been suggested as potential therapeutic targets for CLL. Flavopiridol, a pan-CDK inhibitor, has demonstrated safety and efficacy in patients with relapsed CLL, including patients with high-risk cytogenetic features. In this study, we assessed the therapeutic potential of a covalent CDK7 inhibitor, THZ1, in preclinical CLL models. We observed that THZ1 had a significantly lower IC50 in CLL cell lines MEC1 and MEC2 (7.23nM and 7.35nM, respectively), which was 10-fold more potent than flavopiridol and CR8, two other CDK inhibitors that were previously studied in CLL. THZ1 inhibited cell growth and induced apoptosis in MEC1, MEC2 and primary CLL cells in both a dose-dependent and time-dependent manner. The induction of apoptosis by THZ1 in MEC1, MEC2 and primary CLL cells was associated with PARP1 cleavage and down-regulation of anti-apoptotic protein MCL-1. Particularly, THZ1 was able to overcome protection signals provided by the tumor microenvironments and induce apoptosis in primary CLL cells in stromal-coculture conditions. To elucidate the mechanism of action of THZ1, we performed a time course RNA-seq expression analysis in both MEC1 and MEC2 treated with 50nM THZ1. Based on the incremental reduction of transcript levels over a 12-hour period, we identified 728 and 647 THZ1-sensitive transcripts (~5.2% and 4.5% of all active transcripts) in MEC1 and MEC2 cell lines, respectively. Pathway analysis demonstrated that THZ1 down-regulated transcripts are significantly enriched in regulation of cellular metabolism including hypoxia, mTORC1 signaling, and glycolysis pathways. In particular, when analyzing the entire THZ1 down-regulated expression signature for both MEC1 and MEC2 at 8 hours using Gene Set Enrichment Analysis (GSEA), two of the most significantly enriched gene sets contain gene expression signatures from hypoxia induction and HIF1α target genes. Several of the metabolic transcripts sensitive to THZ1 treatment including SLC2A1 , SLC2A3 , HK2 , ENO2 , and PKFKB4 were validated using quantitative RT-PCR. Immunoblot analysis demonstrated that mTORC1 substrates 4E-BP1 and p70S6K were hypophosphorylated in MEC1 and MEC2 when treated with increasing concentrations of THZ1 over time, indicating the inhibition of mTORC1 activity by THZ1. We also observed that the metabolically sensitive AKT Ser473 phosphorylation had essentially been fully repressed by THZ1 treatment in both MEC1 and MEC2. Furthermore, THZ1 was able to mitigate glucose uptake and reduce lactate production in MEC1 and MEC2 cells. THZ1 also prevented the induction of glycolytic transcripts by hypoxia and hypoxia-mimetic CoCl2 (200μM) treatment in MEC1, MEC2 and primary CLL cells. Analysis of CDK7 ChIP-seq data in Jurkat cells revealed that CDK7 directly binds to the promoter and -24kb, -35kb distal enhancers of SLC2A3 that are known to directly interact with HIF1α, suggesting that CDK7 is likely involved in transcriptional regulation of HIF1α target genes via direct enhancer binding. Experiments are currently underway to determine if CDK7 physically interacts with HIF1α under hypoxia. Overall, we observed that THZ1 could effectively disrupt CLL cell viability and overcome the protective signals present in the leukemia microenvironment by inhibiting the transcriptional activity of HIF1α, which is a key regulator of the interaction of CLL cells with the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of the CDK7 inhibitor as novel therapies for CLL. DisclosuresNo relevant conflicts of interest to declare.

Role of NFAT in Chronic Lymphocytic Leukemia and Other B-Cell Malignancies.

In recent years significant progress has been made in the clinical management of chronic lymphocytic leukemia (CLL) as well as other B-cell malignancies; targeting proximal B-cell receptor signaling molecules such as Bruton Tyrosine Kinase (BTK) and Phosphoinositide 3-kinase (PI3Kδ) has emerged as a successful treatment strategy. Unfortunately, a proportion of patients are still not cured with available therapeutic options, thus efforts devoted to studying and identifying new potential druggable targets are warranted. B-cell receptor stimulation triggers a complex cascade of signaling events that eventually drives the activation of downstream transcription factors including Nuclear Factor of Activated T cells (NFAT). In this review, we summarize the literature on the expression and function of NFAT family members in CLL where NFAT is not only overexpressed but also constitutively activated; NFAT controls B-cell anergy and targeting this molecule using specific inhibitors impacts on CLL cell viability. Next, we extend our analysis on other mature B-cell lymphomas where a distinct pattern of expression and activation of NFAT is reported. We discuss the therapeutic potential of strategies aimed at targeting NFAT in B-cell malignancies not overlooking the fact that NFAT may play additional roles regulating the inflammatory microenvironment.

Targeting Inhibition of N6-Methyladenosine Demethylase Fto Displays Potent Anti-Tumor Activities in Chronic Lymphocytic Leukemia

Introduction Accumulating evidence indicates that Fat mass and obesity-associated protein (FTO), a N6-methyladenosine (m6A) RNA demethylase exerts crucial roles in oncogenesis. FB23-2, a novel inhibitor selectively targeting FTO m6A demethylase activity displayed promising potency in acute myeloid leukemia. Yet, no literature has been reported regarding the effects of FTO and FB23-2 in the tumorigenesis and development of chronic lymphocytic leukemia (CLL). Hence, the aim of this study was to investigate the clinical significance and mechanisms of FTO regulation in CLL. Methods Peripheral blood samples from 55 de novo CLL patients (36 males and 19 females; age range 32-82 years, median 62 years) were collected with informed consents in Shandong Provincial Hospital. CD19+ B cells were isolated with informed consents from healthy donors. Expression levels of FTO mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-seq) were conduct to profile RNA m6A methylation and expression of CLL cells. Lentiviral vectors were constructed to stably silence and overexpress FTO in CLL cells. Besides, cell viability, apoptosis and cell cycle were assessed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. Results Aberrantly increased expression of FTO was observed in CLL patients and CLL cell lines at mRNA and protein level compared with normal B cells from healthy donors (Figure 1A-B). Clinical correlation analyses suggested FTO high expression was significantly associated with 11q23 deletion (p=0.012; Figure 1C). Furthermore, Keplan-Meier plot indicated that elevated FTO expression predicted adverse outcome in CLL patients (HR=1.758, p=0.019; Figure 1D). ROC curve confirmed the prognostic value of FTO in survival of CLL patients (AUC=0.600, p=0.018; Figure 1E). To explore the potential role of FTO in CLL tumorigenesis, CLL cells were transfected with lentiviral vectors to stably silence and overexpress FTO. CLL primary cells and MEC1 cells with silence of FTO exhibited attenuated cell proliferation, increased fast-onset apoptosis (Figure 2A-D). Western blotting assay suggested significant down-regulated Bcl-2, enhanced cleaved-PARP and BAX expression in FTO-deficient CLL cells. Whereas, gain-of-function assay showed promoted cell survival in FTO-overexpressed CLL cells (Figure 2F-G). Additionally, serial dilution of FTO inhibitor FB23-2 decreased viability of MEC1 and primary CLL cells in time-dependent manner, and displayed rare cytotoxicity in normal B cells (Figure 3A-B). Besides, annexin V-PE/7AAD and western blotting assay indicated obvious apoptosis was induced with treatment of FB23-2 in CLL primary cells from 9 de novo CLL patients (Figure 3C-D). Importantly, obvious G2/M phase arrest and enhanced sensitivity to Venetoclax were also detected in FTO-reduced CLL cells. Furthermore, interactive MERIP-seq and RNA-seq of CLL cells with control and deleted FTO expression were performed to investigate the m6A Methylation-Mediated mechanism of FTO regulation of CLL pathogenesis. A total of 573 significantly changed peaks, of which 301 were significantly up-regulated and 272 peaks were significantly down-regulated (Figure 4A). Differentiated peaks were located in 3'UTR (42.58%) and 5'UTR (24.43%). Annotations of bioinformatics analyses indicated that FTO was functionally enriched in cell apoptosis in CLL progression (Figure 4B). Western blotting assay suggested significant down-regulated p-CHK2, c-myc, p-p53, cyclinD1 and enhanced p-H2AX expression in FTO-deficient CLL cells, indicating FTO accelerated CLL cell survival via DNA damage pathway (Figure 4C). Conclusion Taken together, our investigations identified for the first time the oncogenic role of FTO in CLL tumorigenesis and regulatory mechanism of FTO inhibitor FB23-2 in CLL cells by MERIP sequencing and ex vivo evaluation. Expression of FTO was upregulated and associated with inferior prognosis of CLL patients. FB23-2 exerted potent therapeutic potential in abrogating cell survival and inducing cell cycle arrest via m6A methylation. This study provides a rationale on evaluation of FTO-targeted intervention formulating a novel treatment paradigm in progressed CLL that warrants clinical investigation. Disclosures No relevant conflicts of interest to declare.

Targeting Nuclear NOTCH2 by Gliotoxin Recovers a Tumor-Suppressor NOTCH3 Activity in CLL.

NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.

CG-806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broad Signaling Inhibition in Chronic Lymphocytic Leukemia Cells

CG-806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broad Signaling Inhibition in Chronic Lymphocytic Leukemia Cells

Systematic Investigation of Microenvironmental Drug Resistance Mechanisms in Chronic Lymphocytic Leukemia

Introduction and Methods To investigate the mechanisms by which microenvironmental signals induce treatment resistance in chronic lymphocytic leukemia (CLL) we conducted a high-throughput perturbation assay combining pairwise 15 clinically relevant drugs with 18 stimulations mimicking the tumor microenvironment. We combined this with multi-layer omics data using whole exome sequencing, genome-wide DNA-methylation profiles and RNA sequencing to investigate molecular determinants of microenvironmental drug resistance. In parallel we assessed the in-vivo relevance of investigated pathways in 100 CLL infiltrated- and 100 healthy lymph node (LN) biopsies by staining for stimulus specific downstream pathway components using immunohistochemistry (IHC). Results We initially clustered drugs and microenvironmental stimuli separately based on the similarity of their response profiles across all CLL samples. We found drug response profiles to be correlated closely for drugs targeting the same signaling-pathway, e.g. the B-cell receptor inhibitors Ibrutinib (BTK) and PRT062607 (Syk) (Pearson correlation coefficient: r=0.78, 95% CI: 0.72 - 0.83). In contrast, responses to microenvironmental stimuli were less correlated, indicating diverse effects are caused by the tumor microenvironment (e.g. Interleukin 4 (IL-4) vs. Interleukin 2, Pearson correlation coefficient: r=0.17, 95% CI: 0.03 - 0.31). Twelve of the 18 tested stimulations enhanced survival of CLL cells, whereas two reduced the viability of CLL cells in-vitro (comparisons yielded BH adjusted t-test p-values &lt;0.0001). The strongest pro-survival effects were mediated by IL-4 and Toll-like receptor (TLR) 7/8/9 stimulation, whereas transforming growth factor β1 (TGF-β1) and Interleukin-6 reduced the viability of CLL cells in-vitro. Ten of the 18 stimulation effects were modulated by genetic alterations in CLL cells, for 6 stimuli two or more response modulating genetic alterations were identified (FDR 10%). IGHV mutation status for instance modulated the effects of TLR 7/8/9 agonists as well as Interleukine-1β, CD40 ligand and TGF-β1. We will present a detailed analysis of how microenvironmental stimuli depend on specific genetic alterations in CLL. To understand how soluble stimuli interfere with drug response we searched for specific interactions between stimulations and drugs with the following linear model: Viability ~ drug-effect + stimulation-effect + drug-effect:stimulation-effect and tested for significance of the interaction term. This approach allowed us to dissect interactions independently of the individual effects. We discovered different types of drug-effect:stimulation-effect interactions: 1) The microenvironmental stimulus had a strong pro-survival effect on the baseline viability and interfered with the response to drugs (e.g. IL-4:Ibrutinib, p-value&lt; 0.0001). 2) The microenvironmental stimulus had no major effect on baseline viability but significantly interfered with response to drugs (e.g. Interferon-γ:Ibrutinib, p-value: 0.014). 3) The microenvironmental stimulus and the drug exhibited no major individual effect on CLL cell survival, but together significantly increased CLL cell viability (e.g. Interferon-γ:Ralimetinib, p-value: &lt; 0.0001). We will present a detailed analysis of microenvironmental stimuli and their influence on drug response. In the final step we investigated the biological relevance of selected microenvironmental stimuli in patient LN- and bone marrow biopsies by IHC. CLL infiltrated LN exhibited a higher signaling activity of the IL-4 and TLR pathway than non-neoplastic LN (t-test, p-values &lt;0.0001). A high signaling activity of IL-4 was associated with shorter time to treatment, indicating that CLL progresses faster with strong microenvironmental support. Conclusion We present a systematic investigation on the tumor microenvironment in CLL, the underlying molecular determinants as well its interference with drug response which will serve as a scientific resource and starting point for further mechanistical investigations. Figure Disclosures Kriegsmann: Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Sanofi: Research Funding; Morphosys: Research Funding. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees.

The Mithralog EC-7072 Induces Chronic Lymphocytic Leukemia Cell Death by Targeting Tonic B-Cell Receptor Signaling

B-cell receptor (BCR)-dependent signaling is central for leukemia B-cell homeostasis, as underscored by the promising clinical results obtained in patients with chronic lymphocytic leukemia (CLL) treated with novel agents targeting components of this pathway. Herein, we demonstrate that the mithralog EC-7072 displays high ex vivo cytotoxic activity against leukemia cells from CLL patients independently from high-risk prognostic markers and IGHV mutational status. EC-7072 was significantly less toxic against T cells and NK cells and did not alter the production of the immune effector molecules IFN-γ and perforin. EC-7072 directly triggered caspase-3-dependent CLL cell apoptosis, which was not abrogated by microenvironment-derived factors that sustain leukemia cell survival. RNA-sequencing analyses revealed a dramatic EC-7072-driven reprograming of the transcriptome of CLL cells, including a wide downregulation of multiple components and targets of the BCR signaling pathway. Accordingly, we found decreased levels of phosphorylated signaling nodes downstream of the BCR. Crosslinking-mediated BCR activation antagonized CLL cell death triggered by EC-7072, increased the phosphorylation levels of the abovementioned signaling nodes and upregulated BCL2 expression, suggesting that the mithralog disrupts CLL cell viability by targeting the BCR signaling axis at multiple levels. EC-7072 exerted similar or higher antileukemic activity than that of several available CLL therapies and displayed additive or synergistic interaction with these drugs in killing CLL cells. Overall, our findings provide rationale for future investigation to test whether EC-7072 may be a potential therapeutic option for patients with CLL and other B-cell malignancies.

Abstract 2072: Selinexor in combination with chemotherapy or idelalisib elicits a synergistic cytotoxic effect in primary CLL cells

Abstract Despite the therapeutic efficacy of new targeted drugs in chronic lymphocytic leukemia (CLL), treatment of high-risk patients remains an unmet clinical need. Tumor suppressor proteins (TSPs) and growth regulatory proteins (e.g. p53, p21, FoxO3a - the effector of Akt signal transduction, and IκB - the endogenous inhibitor of NF-κB) bind the nuclear export protein XPO1 and are carried through the nuclear pore complex into the cell cytoplasm. XPO1 overexpression plays a pathogenic role in CLL by mislocalizing TSPs and other cargoes in the cytoplasm. Selinexor is an oral inhibitor of XPO1, which is active as single agent in different hematologic malignancies. The purpose of this study was to evaluate the in vitro cytotoxic effects of selinexor, used in combination with chemotherapy drugs or with the PI3K inhibitor, idelalisib, against primary CLL cells. Specifically, this study aimed at identifying combination regimens that might overcome single agent resistance. Purified CLL cells from 29 patients were exposed to selinexor (Sel) in combination with fludarabine (F-ara-A), bendamustine (Ben) or idelalisib (Ide). Drug concentrations and exposure times were selected based on data obtained in an initial cohort of 5 patients. In selected experiments, the M2-10B4 murine stromal cell (SC) line was used in co-culture with CLL primary cells. Cell viability was analysed by Annexin-V/propidium Iodide immunostaining and flow cytometry. Combination index (CI) was determined using Calcusyn software. NF-kB and Akt activity was tested through an ELISA assay. After 72-hour culture, a significant decrease in CLL cell viability was observed when samples were treated with drug combinations (i.e. Sel 0.1 µM + F-ara-A 1 µM/F-ara-A 10 µM/Ben 30 µM/Ben 50 µM/Ide 0.1 µM/Ide 1 µM/ Ide 10 µM; Sel 1 µM + F-ara-A 1 µM/Ben 30 µM/Ben 50 µM) compared to the single compounds. The majority of combinations exerted synergistic cytotoxic effects. Interestingly, the addition of selinexor was effective in restoring the fludarabine sensitivity of CLL cells showing in vitro resistance to fludarabine. Sel was also capable to overcome the SC-mediated drug resistance, as shown by the significantly increased and synergistic cytotoxicity exerted by drug combinations, compared to single agents, toward leukemic cells co-cultured with SC. From the molecular standpoint, preliminary data showed that Sel significantly potentiated the inhibitory effect exerted by single-agent idelalisib on NF-kB and Akt activity. Our data demonstrate that the combination of Sel with chemotherapy or idelalisib has synergistic cytotoxic effects, also counteracting intrinsic or SC-mediated drug resistance. In vivo experiments in the Eμ-TCL-1 mouse model are currently ongoing to further corroborate the efficacy of selected drug combinations. Citation Format: Maria Todaro, Valentina Griggio, Chiara Salvetti, Chiara Riganti, Yosef Landesman, Mario Boccadoro, Candida Vitale, Marta Coscia. Selinexor in combination with chemotherapy or idelalisib elicits a synergistic cytotoxic effect in primary CLL cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2072.

Dissecting the Prognostic Significance and Functional Role of Progranulin in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is known for its strong dependency on the tumor microenvironment. We found progranulin (GRN), a protein that has been linked to inflammation and cancer, to be upregulated in the serum of CLL patients compared to healthy controls, and increased GRN levels to be associated with an increased hazard for disease progression and death. This raised the question of whether GRN is a functional driver of CLL. We observed that recombinant GRN did not directly affect viability, activation, or proliferation of primary CLL cells in vitro. However, GRN secretion was induced in co-cultures of CLL cells with stromal cells that enhanced CLL cell survival. Gene expression profiling and protein analyses revealed that primary mesenchymal stromal cells (MSCs) in co-culture with CLL cells acquire a cancer-associated fibroblast-like phenotype. Despite its upregulation in the co-cultures, GRN treatment of MSCs did not mimic this effect. To test the relevance of GRN for CLL in vivo, we made use of the Eμ-TCL1 CLL mouse model. As we detected strong GRN expression in myeloid cells, we performed adoptive transfer of Eμ-TCL1 leukemia cells to bone marrow chimeric Grn−/− mice that lack GRN in hematopoietic cells. Thereby, we observed that CLL-like disease developed comparable in Grn−/− chimeras and respective control mice. In conclusion, serum GRN is found to be strongly upregulated in CLL, which indicates potential use as a prognostic marker, but there is no evidence that elevated GRN functionally drives the disease.

Non-genotoxic MDM2 inhibition selectively induces a pro-apoptotic p53 gene signature in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous hematologic malignancy. In approximately 90% of cases the TP53 gene is in its wildtype state at diagnosis of this malignancy. As mouse double-minute-2 homolog (MDM2) is a primary repressor of p53, targeting this protein is an attractive therapeutic approach for non-genotoxic reactivation of p53. Since the discovery of the first MDM2 inhibitor, Nutlin-3a, newer potent and bioavailable compounds have been developed. In this study we tested the second-generation MDM2 inhibitor, RG7388, in patient-derived CLL cells and normal cells, examining its effect on the induction of p53-transcriptional targets. RG7388 potently decreased viability in p53-functional CLL cells, whereas p53-non-functional samples were more resistant to the drug. RG7388 induced a pro-apoptotic gene expression signature with upregulation of p53-target genes involved in the intrinsic (PUMA, BAX) and extrinsic (TNFRSF10B, FAS) pathways of apoptosis, as well as MDM2. Only a slight induction of CDKN1A was observed and upregulation of pro-apoptotic genes dominated, indicating that CLL cells are primed for p53-dependent apoptosis. Consequently, RG7388 led to a concentration-dependent increase in caspase-3/7 activity and cleaved poly (ADP-ribose) polymerase. Importantly, we observed a preferential pro-apoptotic signature in CLL cells but not in normal blood and bone marrow cells, including CD34+ hematopoietic cells. These data support the further evaluation of MDM2 inhibitors as a novel additional treatment option for patients with p53-functional CLL.

Abstract 3905: Strategic combination of the cyclin-dependent kinase inhibitor CYC065 with venetoclax to target anti-apoptotic proteins in chronic lymphocytic leukemia

Abstract CYC065 is a cyclin-dependent kinase (Cdk) inhibitor that is highly selective towards Cdk2 and Cdk9. In chronic lymphocytic leukemia (CLL), a disease that is addicted to the over-expression of anti-apoptotic proteins for survival, inhibition of Cdk9 by CYC065 reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription. These actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1, but not Bcl-2, and induced apoptosis in CLL cells in vitro. The IC50 for CYC065-induced CLL cell death after a 24-hr incubation was 0.8 μM, a concentration that is achievable in the clinic at tolerated doses. CYC065 killed the CLL cells equally efficiently in the presence or absence of the human stromal cell line, StromaNKtert, and with or without a stimulation condition that mimics the lymphoid tissue microenvironment (anti-IgM, anti-CD-40, IL-4). Venetoclax, which specifically inhibits Bcl-2 function, is approved for treatment of CLL with del(17p); however upregulation of Mcl-1 is associated with resistance to venetoclax in the lymph nodes. Therefore, we hypothesized that the combination of CYC065 with venetoclax would target the parallel mechanisms that promote the survival control in CLL cells, and induce synergistic cell death by apoptosis. A time course study of the single agents showed that under conditions that mimic the lymph node microenvironment, cell death induction by venetoclax required 6-8 hr to reach the plateau of cell killing and maximal killing by CYC065 occurred after 24 hr, consistent with the different mechanisms of action of the two compounds. Following the removal of CYC065 or venetoclax after 4, 8, 12, or 24 hr incubations, there was no evidence for additional cell death after an additional 48 hr in drug-free medium regardless of the duration of drug incubation. Immunoblots showed recovery of RNA pol II phosphorylation, and restored Mcl-1 expression upon washout of CYC065. The reversible action of these compounds has potential implications for clinical scheduling combining these compounds. Median effect analysis indicated that CYC065 and venetoclax combined synergistically in CLL samples with or without 17p deletion. A dose reduction analysis confirmed mutual potentiation of each other when combined. Combination of IC50 concentrations of CYC065 and venetoclax for 24 hr was sufficient to decrease the viability of CLL cells by over 90% in the lymph node mimicking microenvironment. Thus, these data provided rationale for clinical combination of CYC065 and venetoclax in CLL. CYC065 is currently in a Phase I clinical trial in patients with advanced solid tumors (NCT02552953) using an intermittent dosing regimen which causes at least 24 hr Mcl-1 downregulation in patient PBMCs at well tolerated dose levels. Citation Format: Rong Chen, Yuling Chen, Sheelagh Frame, David Blake, William G. Wierda, Daniella Zheleva, William Plunkett. Strategic combination of the cyclin-dependent kinase inhibitor CYC065 with venetoclax to target anti-apoptotic proteins in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3905.