Killing Of Chronic Lymphocytic Leukemia Cells Research Articles

Combinatorial Treatment of Chronic Lymphocytic Leukemia with Ibrutinib and Venetoclax Is Superior to Treatment with Single Agents in the TCL1 Mouse Model

IntroductionTargeted therapy with the BTK inhibitor ibrutinib or the BH3-mimetic venetoclax has revolutionized the treatment of chronic lymphocytic leukemia (CLL). These inhibitors have a distinct mechanism of action: ibrutinib impacts BCR signaling through BTK inhibition, resulting in displacement of CLL cells from the protective lymph node environment and inhibition of proliferation whereas venetoclax directly kills CLL cells directly by inhibiting anti-apoptotic BCL-2.While both ibrutinib and venetoclax are effective treatments for CLL as single agents, it may be beneficial to combine these treatments. There are indications that cell killing by venetoclax may not be as effective in the lymph node micro-environment due to local upregulation of BCL-XL and MCL-1 [Thijssen et al. Haematologica 2015]. Ibrutinib displaces CLL cells to the blood but does not kill them. Neither agent is curative and both are taken until resistance/relapse occurs, or until treatment is interrupted due to toxicities. Although clinical trials combining these 2 agents are currently underway, their synergistic effects are still largely hypothetical. Therefore we sought to investigate the merits of combining venetoclax with ibrutinib in the murine TCL1 transgenic model for CLL.MethodsIn the present study, immune competent C57BL/6J mice received an adoptive transfer (AT) of TCL1 splenocytes, obtained from full leukemic TCL1 transgenic mice with the same background. Three weeks after AT, when CD5/CD19% reached >90% of all B cells, mice were treated for 4 weeks with venetoclax, ibrutinib or a combination of ibrutinib and venetoclax. Ibrutinib was provided in the drinking water at a concentration of 0.16 mg/ml. Venetoclax was administered daily intra-gastric, at a dose of 100 mg/kg. CLL development was followed by flow cytometry measurements in the peripheral blood.ResultsWithin the first 5 days of treatment, mice receiving ibrutinib single agent showed a rapid transient lymphocytosis (mean WBC numbers at start of treatment: 2.03*107/mL, peak mean at day 2 of treatment: 1.2*108/mL), followed by a reduction in white blood cell (WBC) numbers. Control mice experienced a rapid progression of disease, reaching a mean WBC count of 2.18*108/mL when they had to be sacrificed due to overt leukemia, 8 weeks following AT. Venetoclax and the combination treatment resulted in rapid reductions in WBC count (Figure 1). The maximum drop of WBC counts in the 3 treatment arms as compared to baseline were as follows: ibrutinib 50% at week 6, venetoclax 70% at week 4, combination: 91% at week 5, p<0.001). The mice receiving either monotherapy showed signs of relapse after 4 weeks of treatment, which was manifested by an increase in WBC counts, whereas the combination treatment did not relapse in this timeframe.Spleen sizes of all mice at end of treatment were compared with control mice at week 5. Spleen sizes were significantly reduced in both monotherapies (% reduction: ibrutinib 47%, venetoclax 44%) and even further reduced in mice that received combination therapy (% reduction: 84%) (Figure 2). Untreated mice displayed a disturbed splenic architecture with massively enlarged marginal zones and a loss of demarcation between B and T cells, which was partially restored only in combination treatment.The percentage of infiltrating CLL cells in the spleen was not significantly altered in the monotherapies (67% and 66% of ibrutinib and venetoclax respectively), but was significantly lower in combination treated mice (45%, p<0.001). Number of proliferating splenocytes was only reduced in mice treated with ibrutinib (28%), while cleaved caspase-3 positive B lymphocytes levels, a marker for apoptosis, was only increased in mice treated with venetoclax.Ongoing experiments are aimed at determining levels of BCL-2 and BH3-only members, and functionality of BCR signaling in correlation with the observed effects. Furthermore, we are currently confirming whether the combination results in sustained response compared to the single treatments.ConclusionsIn conclusion, these first data from the TCL1 CLL model support the combination treatment of venetoclax and ibrutinib. Even in this short treatment timeframe, there is a more durable response with the combination treatment, which may be due to the elimination of reservoirs of CLL cells where resistance to single agent treatment can develop. [Display omitted] DisclosuresSlinger:Johnson & Johnson: Research Funding; Abbvie: Research Funding. Balasubramanian:Janssen Research & Development: Employment, Equity Ownership. Eldering:Roche: Research Funding; Gilead: Research Funding; Celgene: Research Funding. Kater:Johnson & Johnson: Research Funding; Abbvie: Research Funding; Celgene: Consultancy, Research Funding.

Apoptosis Resistance and NOTCH1 Mutations Impair Clinical Outcome in Chronic Lymphocytic Leukemia (CLL) Patients Treated with Ibrutinib

Ibrutinib, an oral, selective inhibitor of Bruton's tyrosyne kinase (BTK), demonstrated superior overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) compared with chemoimmunotherapy in CLL both in first line and in relapsed/refractory patients (Byrd, 2015). Moreover, no significant difference was observed for PFS across most genomic subtypes and PFS was significantly better for ibrutinib-treated patients in first/second line vs later lines of therapy (Brown, 2017). However, the mechanisms underlying ability of ibrutinib to kill CLL cells are not well understood. Deng et al (2017) found that BTK inhibition enhances mitochondrial bcl-2 dependence and therefore the basal level of apoptosis measured through bax/bcl-2 ratio before therapy may be important to test sensitivity to ibrutinib. Even more, NOTCH1 mutations (M) trigger apoptosis resistance (Rosati, 2009) and thus may confer poor response to ibrutinib. The primary aims of our research were: 1) to verify the correlations of bax/bxcl-2 and NOTCH1 M with other biological and clinical prognosticators in patients treated with ibrutinib; 2) to evaluate the impact of bax/bcl-2 and NOTCH1 M on ORR, PFS and OS; 3) to confirm bax/bcl-2 and NOTCH1 M as independent prognostic factors. Therefore, we evaluated the efficacy of ibrutinib, in a real-life contest, on 72 patients, median age 66 years (42-85), median number of previous regimens 2 (0-4; 13.9% previously untreated). Patients received 420 mg oral ibrutinib once daily until progression or occurrence of unacceptable side effects. Median follow up on ibrutinib was 12 months. ORR was 91% [complete response (CR): 20%, partial response (PR): 42%, PR with lymphocytosis (PR-L): 29%]. The estimate 1-year PFS and OS were 70% and 72%, respectively. Nineteen patients (26%) discontinued ibrutinib therapy due to progression (n= 8), adverse events (n=9) or due to other reasons (n=2). Five out of 6 patients with Richter's syndrome (RS) had baseline del(17p)/ TP53 mutations (M). PFS was significantly better for patients in first/second line vs later lines of therapy (p=0.045). Bax/bcl-2 was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold was set at the median value >1.5 (range 0.44-5.10). The presence of NOTCH1 M was investigated with ARMS PCR for c.75447545delCT and by Sanger sequencing of NOTCH1 exon 34. Eighteen patients were NOTCH1 M (25%) and 42 had bax/bcl-2<1.5 (58.3%). All 6 patients who underwent Richter's syndrome had bax/bcl-2 <1.5 (p=0.030). NOTCH1 M were strongly correlated with trisomy 12 (10/18; p=0.00002) and with CD49d >30% (16/18; p=0.024). There was no significant correlation between NOTCH1 M and bax/bcl-2 as well as between bax/bcl-2 and del(17)p/ TP53 M . With regard to clinical outcome, lack of response/progression was significantly correlated both with NOTCH1 M (p=0.009) and bax/bcl-2<1.5 (p=0.017), but not with del(17)p/ TP53 M (p=0.09). Significant shorter PFS was observed in NOTCH1 M patients (42% vs 76% at 24 months, p=0.002, Figure) and in bax/bcl-2 ratio<1.5 cases (55% vs 91% at 24 months, p=0.006, Figure). On the contrary, del(17)p/ TP5 M patients did not show significant worse PFS (p=0.26). Moreover, patients with lower bax/bl-2 showed significant shorter OS (63% vs 91% at 24 months, p=0.020). Interestingly, NOTCH1 M and bax/bcl-2 showed synergistic prognostic properties, since NOTCH1 M and bax/bcl-2<1.5 identified a subset at worse prognosis with regard to PFS (43% vs 86% at 24 months, p=0.001, Figure). In our hands, bax/bcl-2 and NOTCH1 M were powerful prognosticators for patients treated with ibrutinib, showing additive clinical effects. In multivariate analysis of PFS, bax/bcl-2 (HR: 0.1; p=0.03) together with NOTCH1 (HR:5.0; p=0.007), were confirmed as independent prognostic factors. Therefore, bcl-2 antagonists, such as venetoclax, could be combined with BTK inhibitors, in order to overcome apoptosis resistance in patients treated with ibrutinib, particularly within the CLL subset characterized by lower bax/bcl-2 ratio and/or NOTCH1 M. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Antihistamines are synergistic with Bruton’s tyrosine kinase inhibiter ibrutinib mediated by lysosome disruption in chronic lymphocytic leukemia (CLL) cells

Lysosomes in chronic lymphocytic leukemia (CLL) cells have previously been identified as a promising target for therapeutic intervention in combination with targeted therapies. Recent studies have shown that antihistamines can induce lysosomal membrane permeabilization (LMP) in a variety of cell lines. Furthermore, our previous data indicates that lysosomotropic agents can cause synergistic cell death in vitro when combined with some tyrosine kinase inhibitors (TKI). In the current study, we have shown that three over-the-counter antihistamines, clemastine, desloratadine, and loratadine, preferentially induce cell death via LMP in CLL cells, as compared to normal lymphocytes. We treated primary CLL cells with antihistamines and found clemastine was the most effective at inducing LMP and cell death. More importantly, the antihistamines induced synergistic cytotoxicity when combined with the tyrosine kinase inhibitor, ibrutinib, but not with chemotherapy. Moreover, the synergy between clemastine and ibrutinib was associated with the induction of reactive oxygen species (ROS), loss of mitochondrial membrane potential and decreased Mcl-1 expression leading to apoptosis. This study proposes a potential novel treatment strategy for CLL, repurposing FDA-approved allergy medications in combination with the targeted therapy ibrutinib to enhance drug efficacy.

Ex Vivo Drug Sensitivity Screens Identify Personalized Treatment Options for CLL Patients

In the era of targeted therapies, it remains a challenge to determine the optimal treatment for the individual cancer patient. By screening the tumor cells for drug sensitivity ex vivo it is possible to predict to which drugs and drug combinations the cancer will respond. We have developed an ex vivo culture protocol that mimics the chronic lymphocytic leukemia (CLL) tumor microenvironment and allows cell proliferation. Using our unique method, we performed drug sensitivity screens on B cells from 22 CLL patients and 5 matched healthy donors. The drug library consisted of 70 single drugs and 39 drug combinations that are clinically relevant for treatment of CLL, including venetoclax (Bcl-2 antagonist), ibrutinib (Btk inhibitor) and idelalisib (PI3K inhibitor). B cells were cultured in 384-well plates in the presence of drugs covering a concentration range of 5 log10 increments. Drug sensitivity was assessed after three days using the CellTiter-Glo luminescent cell viability assay. The Drug Sensitivity Score (DSS) was then calculated for each treatment based on area under the concentration-response curve. We observed specificity towards CLL cells for drugs including TAK-659 (SYK inhibitor), TGR-1202 and duvelisib (PI3K inhibitors), trametinib (MEK1/2 inhibitor) and venetoclax. Ten combinations with venetoclax were investigated, and all showed high drug sensitivity and specificity towards CLL cells. When ranking the drug combinations from high to low DSS, CLL selectivity and obtained synergy, combinations of venetoclax with trametinib, ibrutinib or duvelisib showed the highest potency. Of interest, similar effects were not observed for venetoclax in combination with other Btk or PI3K inhibitors. In order to understand the mechanisms underlying these differences, signaling analyses by use of phospho flow is currently ongoing. In summary, by applying drug sensitivity screens, we have identified drugs and drug combinations that kill CLL cells. These findings should be investigated further in animal models and in precision medicine clinical trials. Disclosures Tjønnfjord: Mundipharma: Research Funding.

Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib Show Expansion of T-Cell Clonotypes Composed of Antitumor Cytotoxic CD8+ T-Cells

We recently showed that ibrutinib induced a more oligoclonal T-cell receptor (TCR) repertoire in patients with chronic lymphocytic leukemia (CLL). Using next generation sequencing (NGS), we found that overrepresented TCRβ clonotypes were mostly patient-specific and that the degree of oligoclonal expansion correlated with CD8+ T-cell counts. Here, we tested the hypothesis that overrepresented clonotypes are CD8+ T-cells that respond to tumor antigens. Flow cytometric profiling of TCR Vβ (TRBV) chain usage, T-cell subset distribution, and granzyme B (GrB) expression was performed on cryopreserved peripheral blood mononuclear cells obtained at baseline and during response to single-agent ibrutinib (n = 5). Antibodies against TRBV were selected to investigate the ten most abundant TCRβ clonotypes identified by NGS. The cumulative frequency of these clonotypes evaluable by flow cytometry increased at the time of response (mean 31.2% ± SEM 3.9% of all T-cells) compared to baseline (mean 24.1% ± SEM 2.3% of all T-cells). Within each TRBV subpopulation, the proportion of CD4+ T-cells decreased (mean fold-change 0.78 ± SEM 0.04), while the proportion of CD8+ T-cells increased (mean fold-change 1.79 ± SEM 0.36). We further observed that the TRBV clonotypes expanding the most during treatment were comprised exclusively of CD8+ T-cells in every patient. These highly expanded CD8+ clonotypes expressed GrB, consistent with a cytotoxic phenotype. To evaluate whether expanding CD8+ clonotypes are tumor-specific, T-cells were expanded ex vivo in two conditions: (1) co-culture with autologous CLL cells, CD40L, IL-2, and IL-7 or (2) with anti-CD3/CD28/CD137 beads, IL-2, and IL-7. Overall, T-cells expanded with autologous CLL cells had a higher proportion of CD8+ GrB+ cells than T-cells expanded with anti-CD3/CD28/CD137 beads. The dominant CD8+ clonotypes detected in each patient at the time of response were preferentially expanded by co-culture with autologous CLL cells compared to expansion with beads. The biased expansion of cytotoxic T-cell clonotypes in the CLL-primed T-cell product supports a TCR mediated response to tumor antigens. Next, cytotoxicity assays were performed by mixing the expanded T-cell products described above with autologous CLL cells at an effector to target ratio of 3:1, 1:1 and 1:3. CLL-primed T-cells killed CLL cells in a dose-dependent fashion. In contrast, bead-expanded T-cells were ineffective at killing CLL cells or even protected tumor cells from spontaneous apoptosis. Thus, tumor-specific cytotoxic T-cells expand during treatment with ibrutinib and are further enriched ex vivo by co-culture with autologous CLL cells. In summary, increased TCR oligoclonality in patients responding to ibrutinib is driven by the expansion of specific CD8+ clonotypes. These clonotypes expand in co-culture with, and are cytotoxic against, autologous CLL cells. Tumor-specific CD8+ T-cell clonotypes may contribute to the overall treatment response with ibrutinib and supports the investigation of therapeutic strategies combining ibrutinib with T-cell directed immunotherapy. Disclosures Baskar: NIH: Patents &amp; Royalties: ROR1 mAb 2A2. Wiestner:Nurix: Research Funding; Acerta: Research Funding; Merck: Research Funding; Pharmayclics: Research Funding.

First Line Treatment with Venetoclax and Ibrutinib Induction Followed By Obinutuzumab Intensification Exclusively in CLL/SLL Patients Not in Complete Remission and/or with Detectable Bone Marrow Minimal Residual Disease (NEXT STEP trial)

First Line Treatment with Venetoclax and Ibrutinib Induction Followed By Obinutuzumab Intensification Exclusively in CLL/SLL Patients Not in Complete Remission and/or with Detectable Bone Marrow Minimal Residual Disease (NEXT STEP trial)

Neddylation Pathway Regulates Treg Differentiation and T Cell Function in Chronic Lymphocytic Leukemia (CLL) Ex Vivo and Murine In Vivo Studies

Introduction: Inhibitors of B-cell receptor associated kinases (e.g., Bruton tyrosine kinase and phoshpoinotiside-3 kinase) have led to significant improvement in outcomes of patients with CLL. Furthermore, such therapies have immunomodulatory effects. This is particularly relevant given that T cells from patients with CLL are functionally compromised and demonstrate impaired immune synapse formation, adhesion, migration and cytotoxicity. It is critical to improve understanding of the immunomodulatory properties of the novel agents as this will help understand their effect on the immune system in CLL, mitigate toxicities as well as inform future drug development. Pevonedistat, (MLN4924) forms an irreversible covalent adduct with NEDD8, a ubiquitin-like modifier, thereby inhibiting the NEDD8-activating enzyme (NAE). This leads to decreased neddylation and reduced activity of cullin-RING E3 ubiquitin ligases (CRLs). Ultimately, a decrease in CRL activity leads to reduced ubiquitination and proteasomal degradation of CRL substrates, extending the half-life of these proteins, including inhibitor of NFκB (IκB). We have shown that pevonedistat abrogates NFκB activation in CLL cells. Importantly, NFκB activation is indispensable in T-cell activation. However, there is paucity of data regarding the immune effects of targeting neddylation. Here we address this knowledge gap and demonstrate that NAE inhibition may have favorable immunomodulatory effects in CLL. Methods: Peripheral blood mononuclear cells were isolated from patients with CLL and T cells were purified using Dynabeads. Pevonedistat was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). For gene expression analysis, FACS-sorted naïve CD4+ T cells were pre-treated with pevonedistat for 1 hour prior to T-cell receptor (TCR; αCD3/CD28) stimulation; RNA was harvested 3 or 24 hours after stimulation and analyzed on a Clariom S microarray chip. For polarization assays, FACS-sorted naïve CD4+ T cells were TCR-activated for 7 days under Th1/2/17/Treg-differentiation conditions. BALB/c mice were administered 60 mg/kg pevonedistat SC twice weekly for 3 weeks and T cell populations were analyzed by flow cytometry. Results: In vitro treatment of T cells with pevonedistat led to rapid reduction in neddylated cullins and stabilization of pIκBα. NAE inhibition did not impede proximal TCR signaling following TCR stimulation (pZAP70, pERK). GSEA demonstrated downmodulation of NFκB and IL-2 signaling pathways in pevonedistat-treated cells by 3 h. Despite this, CD4/CD8+ T cells exhibited normal induction of early activation markers (CD40L, CD69). By contrast, we observed reduced expression of CD38, HLA-DR and PD-1 and diminished CD25 following continuous treatment with pevonedistat for 72 h. This was accompanied by dose-dependent decrease in IL-2 secretion and reduced proliferation of the CD4/CD8+ T cell subsets (CFSE), but no apoptosis. Sorted naive T cells treated with pevonedistat in Th/Treg polarizing conditions exhibited an increase in IFNγ secretion and a decrease in IL-4, suggesting a shift toward Th1 phenotype. Furthermore, we observed a robust decrease of the inducible Treg (iTreg) FoxP3+ population. Loss of iTregs was accompanied by ablated IL-2/STAT5 signaling. Concurrently, we observed a modest increase in Th17 subpopulation following NAE inhibition. We found increased expression of HIF-1α, a CRL target, in pevonedistat-treated T cells, which may have contributed to this phenomenon. To mimic the clinical pharmacokinetics of pevonedistat, we performed 2 h pulse treatment with pevonedistat prior to TCR stimulation. Under these conditions NFκB activity fully recovered by 24 h. Importantly, allogeneic (OCI-LY19 cells) and autologous (CD40L-stimulated CLL cells) T-cell cytotoxicity, perforin and granzyme B production were not disrupted by NAE inhibition. In vivo administration of pevonedistat in immunocompetent BALB/c mice resulted in a decrease of Treg population, confirming in vitro data. Conclusions: Our data suggest that targeting neddylation may help rebalance T cells towards healthy immune subsets in CLL via the reduction of the Treg/Th2 phenotypes. Combined with our earlier reports that targeting NAE kills CLL cells under lymph node-mimicking conditions, these data provide a strong rationale for continued investigation of pevonedistat in CLL and lymphoid malignancies. Disclosures Berger: Millennium Pharmaceuticals, Inc., Cambridge, MA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Danilov:TG Therapeutics: Consultancy; Takeda Oncology: Research Funding; MEI: Research Funding; Verastem Oncology: Consultancy, Other: Travel Reimbursement , Research Funding; AstraZeneca: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Seattle Genetics: Consultancy; Gilead Sciences: Consultancy, Research Funding; Bristol-Meyers Squibb: Research Funding; Aptose Biosciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Celgene: Consultancy; Curis: Consultancy; Abbvie: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy.

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.

MicroRNA dysregulation and multi-targeted therapy for cancer treatment.

We established that loss of miR-15a/16-1 genes on chromosome 13q14 is the most common alteration in Chronic Lymphocytic Leukemia (CLL) and that miR-15/16 are crucial negative regulator of BCL-2, an antiapoptotic gene overexpressed in most CLLs and in many other malignancies. We have also shown that miR-15/16 target ROR1, a cell surface receptor for Wnt5a which can enhance growth/survival of CLL cells. Interestingly, ROR1 is expressed by many cancers, but not by normal adult tissues. Moreover, Venetoclax, the anti-Bcl-2 drug, and Cirmtuzumab, the monoclonal antibody against ROR1, are synergistic in killing CLL cells. Since an additional miR-15/16 locus exists on chromosome 3q25 (miR-15b/16-2), we generated a knocked out mouse model to study its the role in cancer. We observed that the KO mice developed predominantly CLL. Thus, we generated a double knock out mouse model where both miR-15/16 loci were deleted. Surprisingly we observed that 77% of double KO mice developed Acute Myeloid Leukemia (AML). Based on these evidences, we anticipate that also AMLs with low miR-15/16 expression, overexpression of BCL2 and expression of ROR1, would show an excellent response to a combination therapy with Venetoclax and Cirmtuzumab, since both drugs target the same malignant cells that have lost miR-15/16.

Targeting IRAK4 disrupts inflammatory pathways and delays tumor development in chronic lymphocytic leukemia

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88-mutated and -unmutated cases. In the Eµ-TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

PS1145 TARGETED ACTIVATION OF THE PROTEIN PHOSPHATASES SHP-1 AND PP2A ABROGATES CONSTITUTIVE ACTIVATION OF SURVIVAL PATHWAYS IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA (LGLL)

Background: Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Aims: Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. Methods: TLR stimulation was performed with an agonist mix of Pam3CSK4, HKLM, FSL1 and ODN2006. Cytokine secretion and CLL proliferation were assessed by Luminex system and carboxyfluorescein succinimidyl ester staining, respectively. For in vivo studies, the Eμ-TCL1 adoptive transfer mouse model was used. Results: Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88-mutated and –unmutated cases. In the Eμ-TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Summary/Conclusion: Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

PS1144 TARGETING IRAK4 DISRUPTS INFLAMMATORY PATHWAYS AND DELAYS TUMOR DEVELOPMENT IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Interleukin‐1 receptor‐associated kinase 4 (IRAK4) plays a critical role in Toll‐like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease.Aims:Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL.Methods:TLR stimulation was performed with an agonist mix of Pam3CSK4, HKLM, FSL1 and ODN2006. Cytokine secretion and CLL proliferation were assessed by Luminex system and carboxyfluorescein succinimidyl ester staining, respectively. For in vivo studies, the Eμ‐TCL1 adoptive transfer mouse model was used.Results:Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose‐dependent manner. We further observed a decrease in NF‐κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88‐mutated and –unmutated cases. In the Eμ‐TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells.Summary/Conclusion:Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

The Fully Human Anti-CD47 Antibody SRF231 Has Dual-Mechanism Antitumor Activity Against Chronic Lymphocytic Leukemia (CLL) Cells and Increases the Activity of Both Rituximab and Venetoclax

The Fully Human Anti-CD47 Antibody SRF231 Has Dual-Mechanism Antitumor Activity Against Chronic Lymphocytic Leukemia (CLL) Cells and Increases the Activity of Both Rituximab and Venetoclax

Antibodies conjugated with viral antigens elicit a cytotoxic T cell response against primary CLL ex vivo.

Chronic lymphocytic leukemia (CLL) is the most frequent B cell malignancy in Caucasian adults. The therapeutic armamentarium against this incurable disease has recently seen a tremendous expansion with the introduction of specific pathway inhibitors and innovative immunotherapy. However, none of these approaches is curative and devoid of side effects. We have used B-cell-specific antibodies conjugated with antigens (AgAbs) of the Epstein-Barr virus (EBV) to efficiently expand memory CD4+ cytotoxic T lymphocytes (CTLs) that recognized viral epitopes in 12 treatment-naive patients with CLL. The AgAbs carried fragments from the EBNA3C EBV protein that is recognized by the large majority of the population. All CLL cells pulsed with EBNA3C-AgAbs elicited EBV-specific T cell responses, although the intensity varied across the patient collective. Interestingly, a large proportion of the EBV-specific CD4+ T cells expressed granzyme B (GrB), perforin, and CD107a, and killed CLL cells loaded with EBV antigens with high efficiency in the large majority of patients. The encouraging results from this preclinical ex vivo study suggest that AgAbs have the potential to redirect immune responses toward CLL cells in a high percentage of patients in vivo and warrant the inception of clinical trials.

Abstract 4408: Metabolic alterations induced by ibrutinib in CLL cells as a basis for drug combinations to enhance ibrutinib therapeutic activity

Abstract The use of ibrutinib, which targets the Bruton tyrosine kinase, has significantly improved the therapeutic outcome for patients with chronic lymphocytic leukemia (CLL), especially in cases with chromosome 17p deletion, which usually respond poorly to conventional chemotherapy. However, ibrutinib is not completely curative, and a small portion of CLL patients exhibit resistance. The main objectives of this study were to investigate the effect of ibrutinib on CLL mitochondrial metabolism and to develop novel strategies to enhance the therapeutic activity of ibrutinib. Ibrutinib-induced changes in mitochondrial functions were analyzed by measuring oxygen consumption rate (OCR) as an indicator of mitochondrial respiration and extracellular acidification rate (ECAR) as a parameter of lactate production from glycolysis using an extracellular flux analyzer. In a primary CLL cell-stromal cell co-culture system (which maintains long-term CLL cell viability), ibrutinib induced little change in OCR and ECAR during the first 48-72 h. However, prolonged drug exposure (up to 7 days) in vitro caused a consistent and significant decrease in OCR compared to that in untreated co-cultured CLL cells. Importantly, CLL cells isolated from patients treated with ibrutinib for 7 days also had significantly decreased OCR compared to pre-treatment samples from the same patients, suggesting that the impact of ibrutinib on mitochondrial functions occurred in vivo and was clinically relevant. On western blot analysis, ibrutinib induced decreases in expression of certain respiratory chain components associated with AMPK activation. On electron microscopy, CLL cells treated with ibrutinib in vitro for 6 days exhibited lower numbers of mitochondria than untreated cells, which was confirmed by flow cytometry analysis of cells stained with Mitotracker Green. Functional analysis showed that ibrutinib caused CLL cells to take up less glutamine but did not affect glucose uptake, suggesting that ibrutinib has a preferential impact on glutamine metabolism. On testing the possibility that the metabolic alterations and mitochondrial dysfunction induced by ibrutinib render CLL cells vulnerable to compounds that affect mitochondria or inhibit glutamine metabolism, we found that combinations of ibrutinib with metformin (inhibitor of mitochondrial OXPHOS) or with C968 (glutaminase inhibitor) resulted in synergistic activity against CLL cells and induced massive leukemia cell death compared to either drug alone. Notably, the combination of ibrutinib and C968 was synergistic in killing CLL cells with 17p deletion. Overall, our results suggest that ibrutinib induces metabolic alterations characterized by decreased mitochondrial respiration and attenuated glutamine metabolism. Such metabolic alterations provide a biochemical basis for mechanism-based drug combination to enhance the therapeutic activity of ibrutinib. Citation Format: Helene Pelicano, Li Feng, William G. Wierda, Michael J. Keating, Peng Huang. Metabolic alterations induced by ibrutinib in CLL cells as a basis for drug combinations to enhance ibrutinib therapeutic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4408. doi:10.1158/1538-7445.AM2017-4408

Abstract B21: Novel pateamine A analogs to target pro-survival proteins in chronic lymphocytic leukemia

Abstract Introduction: Dysregulation of protein translation is a common feature of cancer. Because the great majority of oncoproteins turn-over rapidly, thus have an acute need for mRNA translation compared to housekeeping proteins, protein synthesis is a promising avenue to explore for targeted cancer therapy. The marine natural product pateamine A (PatA) inhibits cap-dependent translation initiation by binding to the eukaryotic initiation factor 4A (eIF4A). We have previously synthesized a family of PatA-based small molecules. The lead compound, des-methyl, des-amino pateamine A (DMDAPatA), is more stable and easier to synthesize than the parent natural product, and demonstrated anti-cancer activities in both cell lines and animal models. In this study, we extended the investigations of the activities of DMDAPatA in B cell malignancies, focusing on the primary chronic lymphocytic leukemia (CLL) cells. We propose to target the biological rewiring of the CLL cells, namely the overexpression of short-lived pro-survival proteins that keep these cells from undergoing apoptosis. We hypothesize that a transient inhibition of translation will cause a lethal decrease in those labile pro-survival proteins in CLL cells that will initiate apoptosis. Experimental Procedures: Primary CLL cells were used to screen the PatA analogs for their anti-leukemia activity. Cell viability was analyzed by flow cytometry. Protein translation was measured by tritium labeled leucine incorporation. Protein expression was determined by immunoblotting. Results: Our data showed that DMDAPatA blocked protein synthesis in the CLL cells. This action reduced the short-lived anti-apoptotic proteins Mcl-1 and XIAP, and initiated apoptosis in as short as 4 hr. The levels of the more stable anti-apoptotic proteins Bcl-2 and Bcl-XL were not affected. This is consistent with the selective activity of translation inhibitors toward proteins with rapid turn-over rates. Since DMDAPatA depleted Mcl-1 without affecting Bcl-2, and the Bcl-2 antagonist ABT-199 inhibits Bcl-2 activity but spares Mcl-1, the combination of DMDAPatA and ABT-199 targeted both arms of apoptosis control and killed CLL cells synergistically. Dose reduction index analysis demonstrated mutual potentiation of ABT-199 and DMDAPatA. However, in the presence of human plasma proteins, DMDAPatA lost its potency substantially. A plasma protein binding analysis showed over 99% binding in human plasma, which may hamper its development as a therapeutic agent. To address this issue, we synthesized a new series of PatA analogues with the goal of improving the physical properties and CLL potency. We found that any alteration on the binding domain of PatA, either on eastern half of the macrolide ring or the side chain, resulted in dramatic loss of activity toward the CLL cells. The scaffolding domain on the western side of the ring was more tolerable to modifications which made it feasible to improvement. We proposed to introduce an amino group at either the C2 (alpha-amino variant) or C3 position (beta-amino variant) of the macrolide ring to lower lipophilicity and reduce binding to plasma proteins. This strategy led to the identification of three new leads with potent inhibition of protein synthesis. They exhibited greater cytotoxic potency toward CLL cells than DMDAPatA and lower human plasma protein binding. They also showed better selectivity towards CLL cells over normal lymphocytes compared to the parental compound PatA, demonstrating improved therapeutic index. Conclusions: We have successfully synthesized three new PatA analogs with potent anti-leukemia activities. These compounds hold promise for application to cancers with the right biological context, such as CLL, in which the leukemia cells are addicted to the sustained expression of short-lived oncoproteins for survival. Citation Format: Rong Chen, Mingzhao Zhu, Yuling Chen, Wesley Skillern, Qun Qin, William G. Wierda, Kenneth G. Hull, Daniel Romo, William Plunkett. Novel pateamine A analogs to target pro-survival proteins in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B21.

Bruton's tyrosine kinase inhibition increases BCL-2 dependence and enhances sensitivity to venetoclax in chronic lymphocytic leukemia.

Although the BTK inhibitor ibrutinib has transformed the management of patients with CLL, it does not induce substantial apoptosis in vitro, and as such the mechanisms underlying its ability to kill CLL cells are not well understood. Acalabrutinib, a more specific BTK inhibitor now in development, also appears to be highly effective in CLL, but the connection of its mechanism with CLL cell death is also unclear. Using dynamic BH3 profiling, we analyzed alterations in the function of the mitochondrial apoptotic pathway induced by ibrutinib and acalabrutinib. We studied CLL patient samples treated ex vivo with both drugs, as well as primary samples from CLL patients on clinical trials of both drugs. We found that BTK inhibition enhances mitochondrial BCL-2 dependence without significantly altering overall mitochondrial priming. Enhancement of BCL-2 dependence was accompanied by an increase in the pro-apoptotic protein BIM. In contrast, treatment with the selective BCL-2 inhibitor venetoclax enhanced overall mitochondrial priming without increasing BCL-2 dependence. Pre-treatment of CLL cells with either BTK inhibitor, whether ex vivo or in vivo in patients, enhanced killing by venetoclax. Our data suggest that BTK inhibition enhances mitochondrial BCL2 dependence, supporting the ongoing development of clinical trials combining BTK and BCL-2 inhibition.

Regulation of B-Cell Receptor Signalling By the Tumour Microenvironment in Chronic Lymphocytic Leukemia (CLL) and Its Impact on Adhesion and miRNA Expression

B-cell receptor (BCR) mediated signalling is pivotal to the pathogenesis of chronic lymphocytic leukaemia (CLL), which is broadly split into two major subsets based on the immunoglobulin heavy chain variable (IGHV) gene mutational status. Progressive disease is characterised by unmutated IGHV genes (U-CLL) and more indolent disease by mutated IGHV genes (M-CLL). Antigen/autoantigen engagement is crucial for BCR-driven CLL cell survival, disease progression and resistance to therapy. Drugs that target this pathway are revolutionising the treatment of this disease. However, these inhibitors are not curative and some patients develop resistance to ibrutinib following mutation of the BTK or PLCγ2 genes or for as yet unknown reasons. We have shown that IL-4 pre-treatment induced sIgM expression especially in cases with unmutated IGHV genes, augmented anti-IgM induced signalling and compromised the ability of ibrutinib (1 µM) and idelalisib (1 µM) to inhibit BCR-mediated signalling. Previous reports highlighted a role of miR-155 (Cui Blood 2014) and miR150 (Mraz Blood 2014) in regulating BCR signalling in CLL cells by targeting SHIP1 (negative regulator of BCR signalling) and GAB1 and FOXP1 (positive regulators of BCR signalling) respectively. In this study we investigated the effect of IL-4 on expression of these miRNA and demonstrated that IL-4 augments and inhibits expression of miR155 and miR150 respectively (Figure 1), but had no effect on the house keeping control gene RNU6, demonstrating a mechanism whereby IL-4 augments BCR signalling. Furthermore, since BCR engagement can promote adhesion in CLL cells, we investigated the effect of IL-4 alone and in combination with anti-IgM on integrin expression such as CD49d, CD49e and CD29 and on adhesion. IL-4 pre-treatment augmented CD49d (α4) and CD44 expression and promoted anti-IgM as well as PMA mediated adhesion of CD5\\CD19+ CLL cells to fibronectin. Interestingly in contrast to greater sIgM increases by IL-4 in U-CLL, IL-4 promoted greater anti-IgM-induced adhesion largely in the M-CLL subset. This indicates that IL-4 may have distinct effects in the two subsets.Given our results, we hypothesised that dual inhibition of IL-4 and BCR signalling may promote greater responses in patients. At ASH 2015 we demonstrated that cerdulatinib, a dual JAK/SYK inhibitor, now in phase II clinical trials in CLL, can inhibit IL-4 and anti-IgM/anti-IgD induced signalling at concentrations achievable in patients (Cmin 1μM). We have now extended these studies and shown that cerdulatinib treatment prevented IL-4-induced effects such as increased sIgM expression and subsequent downstream signalling, and prevented downmodulation of CXCR4 and to a greater extent than the specific JAK3 inhibitor tofacitinib. Furthermore, inhibition of JAK1/3 by cerdulatinib also reduced IL-6, IL-10, IL-15, IL-21 and IFNγ-induced signalling, which have all been shown to play a role in CLL biology. More importantly, cerdulatinib killed CLL cells under basal conditions, particularly in cases with progressive disease. However, CLL cells within the patient lymph nodes likely receive signals from the BCR, IL-4 and possibly CD40L, which results in the elevation of pro-survival proteins Mcl-1 and Bcl-XL, providing protection from basal and ibrutinib or venetoclax induced killing. Following simultaneous treatment with bead-immobilised anti-IgM, 10ng/ml IL-4 and 300ng/ml CD40L, cerdulatinib was still able to induce a modest amount of apoptosis, however in combination with venetoclax there was a synergistic increase in killing. Together these data provide evidence of how IL-4 increases BCR signalling by directly modulating miRNA expression, but also additional effects on adhesion which may subsequently retain the tumour cells within the lymph nodes. JAK inhibition may prevent these effects. Therefore, these findings indicate that JAK inhibition in combination with BCR kinase inhibition maybe of value for the treatment of CLL. [Display omitted] DisclosuresConley:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Davies:Bayer: Research Funding; Karyopharma: Honoraria, Research Funding; GSK: Research Funding; Janssen: Honoraria; Pfizer: Research Funding; Mundipharma: Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Travel to scientific conferences, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Travel, accommodation, expenses, Research Funding. Johnson:Celldex Therapeutics: Research Funding. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding. Coffey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Steele:Portola Pharmaceuticals: Honoraria.

Targeting p53-deficient chronic lymphocytic leukemia cells in vitro and in vivo by ROS-mediated mechanism.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. Loss of p53 function in CLL cells due to chromosome 17p deletion or p53 mutations often leads to a more malignant disease phenotype and is associated with drug resistance and poor clinical outcome. Thus, development of novel therapeutic strategies to effectively target CLL cells with p53 deficiency is clinically important. Here we showed that p53-null CLL cells were highly sensitive to ROS-mediated cell killing due to their intrinsic ROS stress. We further demonstrated that a natural compound phenethyl isothiocyanate (PEITC) was able to effectively kill CLL cells with loss of p53, even under the protection of stromal cells. In p53-defficient CLL cells, PEITC induced a rapid depletion of glutathione and a severe accumulation of ROS, leading to massive leukemia cell death in the stromal microenvironment. The drug-induced cell death was associated with a significant decrease of in MCL-1 survival molecule. We further showed that ROS-mediated cell death was the key mechanism by which PEITC induced cytotoxicity, since such cell death could be prevented by addition of antioxidant NAC. Importantly, in vivo study showed that PEITC was able to induce substantial leukemia cell death in mice. Treatment of CLL mice harboring TCL1-Tg:p53−/− genotype with PEITC significantly prolonged the median survival time of the animals. Our study identifies a vulnerability of p53-null CLL cells with high sensitivity to ROS-generating agents, and suggests that PEITC may potentially be useful for clinical treatment of CLL with 17p deletion and p53 mutations.

The BCL2 selective inhibitor venetoclax induces rapid onset apoptosis of CLL cells in patients via a TP53-independent mechanism

AbstractBCL2 blunts activation of the mitochondrial pathway to apoptosis, and high-level expression is required for chronic lymphocytic leukemia (CLL) survival. Venetoclax (ABT-199) is a small-molecule selective inhibitor of BCL2 currently in clinical trials for CLL and other malignancies. In conjunction with the phase 1 first-in-human clinical trial of venetoclax in patients with relapsed or refractory CLL (M12-175), we investigated the mechanism of action of venetoclax in vivo, explored whether in vitro sensitivity assays or BH3 profiling correlated with in vivo responses in patients, and determined whether loss of TP53 function affected responses in vitro and in vivo. In all samples tested, venetoclax induced death of CLL cells in vitro at concentrations achievable in vivo, with cell death evident within 4 hours. Apoptotic CLL cells were detected in vivo 6 or 24 hours after a single 20-mg or 50-mg dose in some patients. The extent of mitochondrial depolarization by a BIM BH3 peptide in vitro was correlated with percentage reduction of CLL in the blood and bone marrow in vivo, whereas the half lethal concentration derived from standard cytotoxicity assays was not. CLL cell death in vitro and the depth of clinical responses were independent of deletion of chromosome 17p, TP53 mutation, and TP53 function. These data provide direct evidence that venetoclax kills CLL cells in a TP53-independent fashion by inhibition of BCL2 in patients and support further assessment of BH3 profiling as a predictive biomarker for this drug.