-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member denotes an abstract that is clinically relevant.
denotes that this is a recommended PHD Trainee Session.
denotes that this is a ticketed session. 1608 Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia
Program: Oral and Poster Abstracts
Session: 618. Acute Lymphoblastic Leukemias: Biomarkers, Molecular Markers and Minimal Residual Disease in Diagnosis and Prognosis: Poster I
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, apoptosis, ALL, Translational Research, Combination therapy, Diseases, drug-drug interactions, Therapies, Lymphoid Malignancies, Biological Processes
Saturday, December 9, 2023, 5:30 PM-7:30 PM Colin Fortner, MSc1*, Maren Wichert2*, Alexandra Niedermayer, MSc2*, Klaus-Michael Debatin, Prof. Dr.2, Lüder Hinrich Meyer, Prof. Dr.2* and Felix Seyfried2*
1Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, AL, Germany
2Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany
T-Cell acute lymphoblastic leukemia (T-ALL) is a disease caused by the malignant transformation of T-cell lineage progenitors. With the use of intensive chemotherapies survival rates have improved, but outcomes particularly of relapsed patients remain poor. In addition, currently used intensive chemotherapies are associated with high rates of treatment-related morbidity and mortality, emphasizing the need to develop new and improved therapies.
The intrinsic apoptosis pathway, one of the key pathways controlling cell death, is dysregulated in many cancers and contributes to leukemogenesis and treatment failure. The main steps of this pathway are controlled by proteins of the B-cell lymphoma (BCL-2) family at the outer mitochondrial membrane. Hence, targeting this pathway by BH3-mimetics has emerged as an effective new treatment strategy in different cancers. Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity.
The aims of this study are to evaluate susceptibilities of T-ALL to inhibitors of BCL-2 family proteins, to identify markers of response, to elucidate mechanisms of action and to assess combination effects.
First, we analyzed the anti-leukemia activities of inhibitors targeting BCL-2 (venetoclax), BCL-XL (A-1331852) and MCL-1 (AZD5991) and of the dual BCL-2/BCL-XL inhibitor AZD4320 in T-ALL cell lines (N=6) and a series of patient-derived xenograft (PDX) samples (N=8). Inhibition of MCL-1 alone was not effective in the cell lines tested (EC50 >1 µM), but heterogeneous sensitivity was found in PDX samples (EC50 <1µM in 3/8 samples). In cell lines, sensitivity for BCL-2 inhibition was exclusively found in a cell line with an early T-cell precursor ALL phenotype (Loucy). Similar to cell lines, we found insensitivity for BCL-2 inhibition in all PDX samples tested (N=8). For inhibition of BCL-XL, we found heterogeneous sensitivities with 4/6 cell lines and 5/8 PDX samples showing an EC50 <1 µM. Interestingly, BH3-profiling confirmed BCL-2 dependence in Loucy and BCL-XL dependence in the other cell lines. Importantly, dual inhibition of BCL-2 and BCL-XL showed clear responses in cell lines and PDX samples (median EC50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (rs=0.89, p=0.03), providing a marker of response.
We next sought to elucidate the molecular mechanisms by which different BH3-mimetics induce cell death in T-ALL. Analyzing protein interactions of BCL-2 family members by co-immunoprecipitation studies, we found that AZD4230 acts on-target by releasing BIM from BCL-2 and BCL-XL, but with compensatory increased protein complexes of BIM and MCL-1. Accordingly, combined inhibition of BCL-2 and BCL-XL with inhibition of MCL-1 leads to release of BIM from all three anti-apoptotic proteins enabling downstream apoptosis signaling. These findings suggest that combined inhibition of BCL-2 and BCL-XL together with MCL-1 may provide synergistic benefits. To further validate these data, we applied dynamic BH3-profiling and determined dependencies of T-ALL samples on BCL-2 family members in response to BCL-2/BCL-XL inhibition. Here, we found that the MCL-1 dependence strongly increases upon BCL-2/BCL-XL inhibition, suggesting MCL-1 as a target for combined inhibition.
Based on these results, we performed dose-response matrix analyses to test the effects of combined BCL-2/BCL-XL and MCL-1 inhibition. Most importantly, combined inhibition showed synergism in all cell lines and PDX samples tested (Bliss scores >10), including leukemias being insensitive to single compounds.
Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.
Disclosures: No relevant conflicts of interest to declare.
-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member denotes an abstract that is clinically relevant.
denotes that this is a recommended PHD Trainee Session.
denotes that this is a ticketed session. 84 Pre-Clinical Study on the Dual BCL2/BCL-XL Inhibitor AZD0466 for the Treatment of Chronic Lymphocytic Leukemia
Program: Oral and Poster Abstracts
Type: Oral
Session: 641. Chronic Lymphocytic Leukemias: Basic and Translational: Molecular Determinants of CLL and Richter Transformation
Hematology Disease Topics & Pathways:
Research, Translational Research, Therapies
Saturday, December 9, 2023: 10:45 AM Billy Jebaraj, PhD1*, Rashmi Priyadharshini Dheenadayalan, PhD2*, Annika Müller, PhD3*, Jialei Qi3*, Eugen Tausch, MD4*, Christof Schneider, MD5*, Daniela Steinbrecher, PhD3*, Daniel Mertens, PhD3*, Felix Seyfried6*, Lüder Hinrich Meyer, Prof. Dr.6*, André Lechel, PhD7*, Barbara F. Eichhorst, MD8, Courtney L Andersen, PhD9*, Giulia Fabbri, MD, PhD10* and Stephan Stilgenbauer, MD1
1Department of Internal Medicine III, Division of CLL, University Hospital Ulm, Ulm, Germany
2University of Ulm, Ulm, Germany
3University Hospital Ulm, Ulm, Germany
4Department of Internal Medicine III, Division of CLL, Ulm University, Ulm, Germany
5Division of CLL, Department of Internal Medicine III, University of Ulm, Ulm, Germany
6Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany
7Ulm University Hospital, Ulm, Germany
8University of Cologne, Cologne, Germany
9AstraZeneca Early Oncology R&D TDE, Waltham, MA
10Oncology R&D AstraZeneca, Waltham, MA
Targeting BCL2 with venetoclax has proven successful for treatment of chronic lymphocytic leukemia (CLL), but resistance develops in a subset of patients. Among other mechanisms, mutations in BCL2 and increased BCL-XL expression confer venetoclax refractoriness in patient subgroups. It is therefore important to identify alternative treatment options targeting BCL-XL and BCL2 for combating venetoclax resistance. AZD4320 was developed as a dual BCL-2/BCL-XL inhibitor. AZD0466 is a dendrimer-drug conjugate of AZD4320, designed to optimize drug release and improve drug tolerability. Hence, we used the nano drug AZD0466 for in vivo experiments and AZD4320 for in vitro and ex vivo experiments.
Firstly, we studied the efficacy of AZD0466 in vivo in the Eµ-TCL1 murine CLL adoptive transfer model. Due to prevalence of primary resistance, murine tumors with the best ex vivo response to AZD4320 were selected and 10 million cells were transferred by i.v. in syngeneic recipient mice. Upon reaching a tumor load of 10% CD19+ CD5+ cells in peripheral blood, mice were randomized and treated with 30mg/kg ibrutinib in drinking water, 34mg/kg or 70mg/kg of AZD0466 once weekly (QW) by i.v, or the vehicle. Treatments were continued until reaching a predefined humane endpoint. AZD0466 at 70mg/kg led to a significant increase in survival (median 72 days vs. 38 days, P=0.0009) similar to ibrutinib (67 days, P=0.0026) when compared to vehicle (Fig. 1). Analysis of blood parameters at euthanasia did not reveal significant difference in platelet counts between AZD0466, ibrutinib and vehicle treatments.
To evaluate if combination treatment of AZD0466 with BTK inhibitors would improve efficacy, we transplanted murine Eµ-TCL1 tumors into syngeneic recipient mice and randomized them for treatment with vehicle, ibrutinib (30mg/kg in drinking water), acalabrutinib (25mg/kg, p.o. QD), AZD0466 (70mg/kg, i.v., QW) and combination of AZD0466 with ibrutinib or acalabrutinib. AZD0466 and vehicle treatments showed similar platelet and RBC counts measured 2 or 3 days after the 2nd and 4th dosing but, a significant increase in platelet and RBC counts were observed with the combination treatments. A significant decrease in WBC count after 2 weeks, and in bone marrow tumor load after 4 weeks was observed with combined AZD0466 and acalabrutinib treatment compared to the single drugs. Though spleen weights did not differ after 4 weeks between vehicle and AZD0466 monotherapy, a strong decrease in spleen weight and tumor cell number in spleen was observed with the combination of AZD0466 with ibrutinib or acalabrutinib compared to vehicle. To validate the findings in human CLL, we performed dynamic BH3 profiling on primary CLL samples with mutated (N=5) and unmutated IGHV (N=7). Exposure of CLL cells to 1µM acalabrutinib for 24 hours significantly increased BCL2 dependency in all CLL samples with unmutated IGHV and in a subset of mutated IGHV samples. These tumors also showed an increased response to venetoclax and AZD4320 upon exposure to acalabrutinib.
To study the efficacy of the dual BCL2/BCL-XL inhibitor in the context of venetoclax resistance, we stably expressed the BCL2 mutations A113G, D103E, D103V, D103Y, G101V, R129L, V156D and wildtype (WT) in the venetoclax sensitive mantle cell lymphoma cell line MAVER-1. Response to AZD4320 or venetoclax was analyzed using Annexin V/7AAD staining and flow cytometry after 48 hours of treatment. The BCL2 mutants showed varying degrees of resistance to venetoclax. MAVER-1 expressing WT had an IC50 of 17.7nM while D103E, D103V, D103Y, G101V showed the strongest resistance to venetoclax with 5, 13.8, 4.5 and 7.3 fold increase in IC50 compared to WT. Of note, compared to WT (IC50 43.9nM), D103E, D103V and D103Y were sensitive to AZD4320 with a fold change in IC50 of 0.6, 1.8 and 1.3, respectively. G101V mutant cells showed a 5 fold increase in IC50 to AZD4320 compared to WT (Fig. 2). Moreover, AZD4320 was highly efficacious in MAVER-1 and MINO cell line models where resistance to venetoclax mediated by BCL-XL upregulation was modelled by an in vitro dose escalation method.
In summary, our pre-clinical study shows that the dual BCL2/BCL-XL inhibitor could represent an important treatment option for venetoclax resistance mediated by specific BCL2 mutations or BCL-XL upregulation and that its efficacy could be further improved upon combination treatment with BTKi.
Disclosures:Tausch:Abbvie: Consultancy, Honoraria, Other: Travel Support, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Other: travel support, Speakers Bureau; BeiGene: Consultancy, Other: Travel support, Speakers Bureau. Schneider:Abbvie: Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; BeiGene: Other: travel support; Jannsen Cilag: Consultancy. Eichhorst:F. Hoffmann-La Roche Ltd: Honoraria, Research Funding, Speakers Bureau; BeiGene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Research Funding; Janssen: Consultancy, Research Funding, Speakers Bureau; Lilly: Consultancy, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria, Research Funding, Speakers Bureau. Andersen:AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Fabbri:AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Stilgenbauer:Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; GSK: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; Sunesis: Consultancy, Honoraria, Other: travel support, Research Funding.
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