Dose-response Matrix Research Articles

Abstract PO4-23-09: Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells

Abstract In HER2-enriched (HER2E) breast cancer (BC), the dual HER2/EGFR inhibitor Lapatinib is a standard of care for advanced staged disease. Anti-HER2 therapies disrupt tumor progression through inhibition of the PI3K/AKT pathway. Some treated tumors will override inhibition by shifting to MAPK/ERK signaling to fuel cell growth, thus fostering resistance to anti-HER2 therapy. The tyrosine receptor kinase FGFR4 is included in the profile for a HER2E subtype. FGFR4 is overexpressed in ~30% of BC and can signal through PI3K/AKT and MAPK/ERK. Inhibitors for FGFR4 such as BLU554 are being investigated in phase I/II clinical trials for hepatocellular carcinoma, showing success in tumors that are positive for FGFR4’s preferred ligand FGF19. According to cBioPortal patient data, FGF19 is amplified in ~20% of BC, and has been shown to trigger an autocrine loop in triple negative BC through FGFR4. We examined BLU554 as a dual therapeutic with Lapatinib and dissected intracellular signaling stemming from the combination in HER2E BC. MDA-MB-453 BC cells (HER2+FGFR4+) were used for a dual strategy with BLU554 and Lapatinib. Cell viability assays were performed at matched doses, then as a dose response matrix, ranging from 0.1-20 µM. Findings were used for synergy analysis using synergyfinder.fimm.fi. Western blots determined protein activity in response to treatments. Ridaforolimus (mTOR inhibitor) was used in combination with either BLU554 or Lapatinib to assess MAPK/ERK and PI3K/AKT pathway crosstalk in MB-453 cells. Additionally, FGF19 was added to cells after a 2-hour pre-treatment to challenge BLU554 + Lapatinib and compare changes in signaling with FGF19 present. MB-453 cells treated with BLU554 decreased cell viability similarly to Lapatinib at 10 µM (65% [SD ± 3.3] and 63% [SD ± 2.1], respectively). Dual treatment decreased viability compared to single agents at all concentrations but the greatest difference was observed at 20 µM (38.5% in dual compared to Lapatinib [p-value = 0.00006]; 45.1% in dual compared to BLU554 [p-value = 0.00016]). Dose-response matrix of dual treatment yielded a ZIP synergy score of 16.852 ± 2.97 (>10 is synergistic), with the greatest synergy and inhibition at 10 µM and 20 µM of both drugs. BLU554 caused a therapeutic potency shift of Lapatinib, such that lower doses of Lapatinib (0.1 µM, 1 µM) are effective at higher doses of BLU554 (10 µM, 20 µM). Westerns of MB-453 with one dose of BLU554 increased phospho-ERK1/2 (pERK1/2) at 16- or 48-hr time points versus control, but when BLU554 was re-dosed then pERK1/2 was successfully inhibited. Both pAKT and pS6RP were increased by re-dosing BLU554. Cells treated for 2 hrs with Lapatinib decreased pAKT and pS6RP and did not change pERK, whereas a 2-hr treatment of BLU554 increased pAKT and decreased pERK. Dual treatment exhibited inhibition of ERK, AKT, 4EBP1, and S6RP phosphorylation all to a greater extent than single agents in combination with Ridaforolimus. Challenge by ectopic FGF19 of BLU554 + Lapatinib was unable to restore downstream signaling of AKT and ERK. BLU554 synergistically interacts with Lapatinib to decrease cell viability while increasing its therapeutic potency. Lapatinib primarily inhibits AKT signaling, whereas BLU554 targets ERK. Dual treatment simultaneously blocks MAPK/ERK and PI3K/AKT signaling and yields decreases of mTOR downstream effectors pS6RP and p4EBP1. These effects are maintained even when challenged with FGF19, suggesting that dual inhibition of FGFR4 and HER2 could prevent pathway activation from external stimuli. Citation Format: Jordan Beardsley, Joseph Goode, Deborah Altomare. Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-09.

Abstract 7089: Proteomic evaluation of combination data in acute myeloid leukemia to inform mechanism of action and new targets

Abstract Background: Acute myeloid leukemia (AML) is a prevalent adult leukemia. In the US, around 20,380 individuals were diagnosed with AML in 2023. Despite standard therapies, relapse and resistance persist. Cyclin-dependent kinase 9 (CDK9) has been recognized an actionable therapeutic target in AML. The potent inhibitor, AZD4573, induces durable apoptosis and suppresses tumor growth. Our study reveals that combining AZD4573 with standard-of-care (SoC) treatments deepens cell death responses in multiple AML cell models. We also used proteomics tools to understand the mechanisms underlying the vulnerability and resistance in SoC and CDK9 combinations. Methods: In the drug combination study, three SoC agents (cytarabine, decitabine, and venetoclax) were evaluated in combination using a 6 × 6 dose-response matrix in four AML cell models (MOLM13, MOLM16, MV411, and OCI-AML2). The HSA synergy score and Emax drug activity were used to assess the benefits of the drug combinations. In the proteomics evaluation study, MOLM13 cells treated for 7 days under different conditions - (1) vehicle control; (2) cytarabine alone; (3) decitabine alone; (4) venetoclax alone; (5) AZD4573 alone; (6) AZD4573+cytarabine; (7) AZD4573+decitabine; (8) AZD4573+venetoclax - were collected for proteomics analysis using LC-MS/MS. Differential expression and missing pattern analyses were used to identify dysregulated proteins. Functional enrichment analysis was employed to identify dysregulated biological processes and pathways in response to different treatments. Results: Our drug combination screens demonstrated the efficacy of AZD4573 in combination with cytarabine, decitabine, and venetoclax in 3 of 4 AML cell models. In our proteomic analysis, we took two approaches. First, we compared the proteomes of AML cells treated with SoC agents, identifying shared downregulated pathways such as serine biosynthesis, notably pointing to the rate-limiting enzyme PHGDH as a potential therapeutic target in AML. Cytarabine and decitabine induced an inflammatory response, while venetoclax increased the expression of cell division regulators like Aurora kinase B (AURKB). Interestingly, targeting AURKB with AZD2811 has been shown to overcome venetoclax resistance in AML. Second, we compared proteome changes induced by various AZD4573 combinations. We identified IRF2BP2 as a shared vulnerable protein across the three AZD4573 combinations. POUF2F2 expression was significantly induced by cytarabine but suppressed when combined with AZD4573. Given that POUF2F2 activates the PDK1-Akt pathway, our findings suggest that AZD4573-induced POUF2F2 downregulation suppresses the PDK1-AKT pathway, inhibiting tumor progression. Conclusions: Our proteomics analysis identifies vulnerable and resistant pathways and proteins, shedding light on the mechanism of action and revealing potential new targets for AML. Citation Format: Lori Chan, Zuleyha Ozen, Anthony Iannetta, Eric Miele, Funda Kar, Andrew Jarnuczak, Geoff Nelson, Aleksandra Markovets, Justine Roderick-Richardson, Jelena Urosevic, Lisa Drew, Jerome Mettetal. Proteomic evaluation of combination data in acute myeloid leukemia to inform mechanism of action and new targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7089.

Abstract 3603: NKX101, an allogeneic off-the-shelf CAR NK cell therapy targeting NKG2D-Ls, has potent anti-leukemic activity alone or in combination with Ara-C

Abstract Introduction: NKG2D-Ligands (NKG2D-Ls) are a family of immunomodulatory proteins that are upregulated on damaged or transformed cells but have limited expression on normal tissues. Natural killer (NK) cells target and eliminate NKG2D-L+ cells through NKG2D receptor binding and subsequent activation. NKX101 is an allogeneic, off-the-shelf healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR and membrane-bound IL-15 for extended persistence. In a clinical trial, NKX101 treatment combined with modified lymphodepletion incorporating cytarabine (Ara-C) has shown promise for the treatment of relapsed/refractory (r/r) AML, including generating complete responses (CR) with MRD negativity (MRD−). To support the rationale of NKX101 combined with modified lymphodepletion, we developed assays to (i) investigate the cytotoxic activity of NKX101 against individual NKG2D-Ls, (ii) determine the effect of Ara-C pre-treatment on NKG2D-L expression on AML cell lines, and (iii) evaluate the combined anti-leukemic activity of NKX101 and Ara-C. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. CHO-K1 cells were engineered to stably express individual NKG2D-Ls or transduced with empty vector controls and were combined with NKX101 cells for cytotoxicity experiments. AML cell lines were treated with increasing levels of Ara-C and evaluated for NKG2D-L expression by flow cytometry. To measure NKX101 cytotoxicity against AML cell lines, NucRed™-labeled target cells were pre-treated with Ara-C using IC50 and IC90 concentrations and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte® instrument for five days. Assessment of the contribution of each agent was determined using a dose-response matrix of increasing concentrations of NKX101 and Ara-C to generate specific cytotoxicity values. Combination effects were analyzed using the SynergyFinder Plus web application. Results: Expression of a single NKG2D-L was sufficient to trigger NKX101 cytotoxicity against target cell lines, further validating NKG2D-Ls as therapeutic targets. Flow cytometry analysis revealed that pre-treatment with Ara-C upregulated NKG2D-Ls on AML cell lines in a dose-dependent manner. NKX101 showed enhanced anti-leukemic activity against AML cells pre-treated with Ara-C compared to untreated controls, demonstrating that the combination can enhance NKX101 potency. Assessment of the combination of NKX101 and Ara-C using a dose-response matrix revealed that the two modalities combine to potently kill AML cells in a synergistic manner, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML. Citation Format: Cynthia Cho, Nikki Kimura, Jessica Sood, Damie Juat, Max Zhang, Carmel Chan, Tao Geng, Kyle Hansen, James Trager. NKX101, an allogeneic off-the-shelf CAR NK cell therapy targeting NKG2D-Ls, has potent anti-leukemic activity alone or in combination with Ara-C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3603.

Two-way pharmacodynamic modeling of drug combinations and its application to pairs of repurposed Ebola and SARS-CoV-2 agents.

Existing pharmacodynamic (PD) mathematical models for drug combinations discriminate antagonistic, additive, multiplicative, and synergistic effects, but fail to consider how concentration-dependent drug interaction effects may vary across an entire dose-response matrix. We developed a two-way pharmacodynamic (TWPD) model to capture the PD of two-drug combinations. TWPD captures interactions between upstream and downstream drugs that act on different stages of viral replication, by quantifying upstream drug efficacy and concentration-dependent effects on downstream drug pharmacodynamic parameters. We applied TWPD to previously published in vitro drug matrixes for repurposed potential anti-Ebola and anti-SARS-CoV-2 drug pairs. Depending on the drug pairing, the model recapitulated combined efficacies as or more accurately than existing models and can be used to infer efficacy at untested drug concentrations. TWPD fits the data slightly better in one direction for all drug pairs, meaning that we can tentatively infer the upstream drug. Based on its high accuracy, TWPD could be used in concert with PK models to estimate the therapeutic effects of drug pairs in vivo.

Interactions of plumbagin with five common antibiotics against Staphylococcus aureus in vitro.

Staphylococcus aureus is the main culprit, causing a variety of severe clinical infections. At the same time, clinics are also facing the severe situation of antibiotic resistance. Therefore, effective strategies to address this problem may include expanding the antimicrobial spectrum by exploring alternative sources of drugs or delaying the development of antibiotic resistance through combination therapy so that existing antibiotics can continue to be used. Plumbagin (PLU) is a phytochemical that exhibits antibacterial activity. In the present study, we investigated the in vitro antibacterial activity of PLU. We selected five antibiotics with different mechanisms and inhibitory activities against S. aureus to explore their interaction with the combination of PLU. The interaction of combinations was evaluated by the Bliss independent model and visualized through response surface analysis. PLU exhibited potent antibacterial activity, with half maximal inhibitory concentration (IC50) and minimum inhibitory concentration (MIC) values against S. aureus of 1.73 μg/mL and 4 μg/mL, respectively. Synergism was observed when PLU was combined with nitrofurantoin (NIT), ciprofloxacin (CPR), mecillinam (MEC), and chloramphenicol (CHL). The indifference of the trimethoprim (TMP)-PLU pairing was demonstrated across the entire dose-response matrix, but significant synergy was observed within a specific dose region. In addition, no antagonistic interactions were indicated. Overall, PLU is not only a promising antimicrobial agent but also has the potential to enhance the growth-inhibitory activity of some antibiotics against S. aureus, and the use of the interaction landscape, along with the dose-response matrix, for analyzing and quantifying combination results represents an improved approach to comprehending antibacterial combinations.

In Vitro Evidence of Synergistically Enhanced Efficacy of Axitinib When Combined with Asciminib in T315I Mutated Chronic Myeloid Leukemia

Background: Development of ABL1 kinase domain (KD) mutation is one of resistance mechanism to ATP-binding pocket inhibitor (ABPI) therapy. T315I mutation in ABL KD confers broad-spectrum resistance to all 1st- and 2nd-generation ABPIs including Imatinib (IMA), Dasatinib (DAS), Nilotinib (NIL) and Bosutinib (BOS). Ponatinib (PON), a 3 rd generation inhibitor, can overcome T315I-mediated resistance in chronic myeloid leukemia (CML) treatment. However, its increased risk of cardiovascular toxicity limits the broader use of PON in the clinic. Asciminib (ASC) is a Specifically Targeting ABL1 Myristoyl Pocket (STAMP) inhibitor and has been approved for CML treatment in the patients who failed at least two previous lines of TKI therapy. ASC, which blocks the myristoylate pocket in ABL1 KD protein, can potentially overcome ABL1 KD mutation (KDM)-mediated tyrosine kinase inhibitor (TKI) resistance by combining with ABPIs which blocks ATP binding pockets in ABL1 KD protein. However, in vitro evidence to support double blockade approach is still to be further investigated including combination with ABPIs as well with other drug compounds. Axitinib (AXI), which is approved for the treatment of advanced renal cell carcinoma (RCC), is an ABPI that can specifically bind to the ATP-binding pocket in ABL1 KD protein structurally harboring the T315I mutation. In the Cancer Therapeutics Response Portal (CTRP) database, which integrates drug compound and cell line experiment databases, AXI showed very low IC 50 in CML cell lines compared to other cancer cell lines, including RCC. The present study attempted to provide in vitro evidence of the synergistic enhancement of therapeutic efficacy of AXI combined with ASC to overcome T315I mutated CML. Methods and Materials: We analyzed IC 50 values for 649 cell lines tested with AXI using the CTRPv2 database. The drug response of AXI in K562/Wild-Type (WT), K562/T315I mut, BaF3/WT, and BaF3/T315I mut cell lines was evaluated using the WST-8 assay. From the measured IC 50 value in those cell lines as the baseline concentration (conc), AXI and ASC conc were serially diluted. The dose-response matrix and ZIP synergy score were analyzed using SynergyFinder. Results We compared the sensitivity of 649 different cell lines to AXI in the CTRPv2 database and confirmed that the IC 50 of AXI in CML (median IC 50 = 0.31 uM) is 96 times lower than that of various cell lines derived from RCC (median IC 50 = 29.85 uM) (Fig. 1A). This result implies that AXI is more effectively inhibit CML cells at a lower conc than RCC cell lines or other cell lines. When measuring the inhibitory activityof AXI (Fig. 1B), AXI treatment showed 2.22 times lower IC 50 in the K562/T315I mut (IC 50 = 111.6nM) than in the K562/WT (IC 50 = 248nM). Also AXI treatment showed 4.73 times lower IC 50 in the BaF3/T315I mut (IC 50 = 84nM) than in the BaF3/WT (IC 50 = 397.4nM). Overall, the T315I carrying CML cell lines were much more sensitive to lower dose AXI than wild type CML cell lines. In the dose-response matrix analysis (Fig. 1C), AXI treatment alone at 100 nM inhibited the growth of the K562/T315 mut cell line by 38.42%, and ASC treatment alone at 100 nM, by 36.52%. Of note, the combination of AXI at 25nM and ASC at 12.5nM inhibited cell growth by 38.1%, and the combination of AXI at 12.5nM and ASC at 25nM showed 38.42% inhibition, which is similar to the inhibition achieved by 100nM of each drug alone. The result suggests that the combination of reduced dose of each drug, by 25% or 12.5% from the baseline IC 50 dose, showed equivalent inhibitor activity to the baseline IC 50 concentration of each drug monotherapy in the K562/T315 mut cell line. Similarly, when AXI and ASC were combined at a reduced concentration by a half or a quarter and tested in the BaF3/T315I mut cell line, a higher inhibitory activity was observed compared to the baseline IC 50 concentration of each drug monotherapy. The ZIP score was calculated as 31.7 and 20.64 in K562/T315 mut and BaF3/T315I mut cell line. Given that ZIP score was above 10 each, synergistic enhancement of inhibitory activity was demonstrated against T315I mutant CML cells between AXI and ASC. Conclusion: This result suggests synergistically enhanced inhibitory activity of dual blockade using AXI combined with ASC specifically for T315I mutant CML. This approach will be promising against CML cells carrying compound mutation with T315I mutation, which is highly resistant even to Ponatinib or Asciminib.

Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia

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 EC 50 <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 EC 50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (r s=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.

Drug combinations screening using a Bayesian ranking approach based on dose-response models.

Drug combinations have been of increasing interest in recent years for the treatment of complex diseases such as cancer, as they could reduce the risk of drug resistance. Moreover, in oncology, combining drugs may allow tackling tumor heterogeneity. Identifying potent combinations can be an arduous task since exploring the full dose-response matrix of candidate combinations over a large number of drugs is costly and sometimes unfeasible, as the quantity of available biological material is limited and may vary across patients. Our objective was to develop a rank-based screening approach for drug combinations in the setting of limited biological resources. A hierarchical Bayesian 4-parameter log-logistic (4PLL) model was used to estimate dose-response curves of dose-candidate combinations based on a parsimonious experimental design. We computed various activity ranking metrics, such as the area under the dose-response curve and Bliss synergy score, and we used the posterior distributions of ranks and the surface under the cumulative ranking curve to obtain a comprehensive final ranking of combinations. Based on simulations, our proposed method achieved good operating characteristics to identifying the most promising treatments in various scenarios with limited sample sizes and interpatient variability. We illustrate the proposed approach on real data from a combination screening experiment in acute myeloidleukemia.

NKX101, an Allogeneic Off-the-Shelf CAR NK Cell Therapy Targeting NKG2D-Ls, Has Potent Anti-Leukemic Activity Alone or in Combination with Ara-C

Introduction NKG2D-Ligands (NKG2D-Ls) are a family of immunomodulatory proteins that are upregulated on damaged or transformed cells but have limited expression on normal tissues. Natural killer (NK) cells target and eliminate NKG2D-L + cells through NKG2D receptor binding and subsequent activation. NKX101 is an allogeneic off-the-shelf healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR and membrane-bound IL-15 for extended persistence. In a clinical trial, NKX101 treatment combined with modified lymphodepletion incorporating cytarabine (Ara-C) has shown promise for the treatment of relapsed/refractory (r/r) AML, including generating complete responses (CR) with MRD negativity (MRD -). To support the rationale of NKX101 combined with modified lymphodepletion, we developed assays to (i) investigate the cytotoxic activity of NKX101 against individual NKG2D-Ls, (ii) determine the effect of Ara-C pre-treatment on NKG2D-L expression on AML cell lines, and (iii) evaluate the combined anti-leukemic activity of NKX101 and Ara-C. Methods NKX101 cells were generated from peripheral blood leukopaks from healthy donors. CHO-K1 cells were engineered to stably express individual NKG2D-Ls or empty vector controls and were combined with NKX101 cells for cytotoxicity experiments. AML cell lines were treated with increasing levels of Ara-C and evaluated for NKG2D-L expression by flow cytometry. To measure NKX101 cytotoxicity against AML cell lines, NucRed™-labeled target cells were pre-treated with Ara-C using IC 50 and IC 90 concentrations and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte® instrument for five days. Assessment of the contribution of each agent was determined using a dose-response matrix of increasing concentrations of NKX101 cells and Ara-C to generate specific cytotoxicity values. Combination effects were analyzed using the SynergyFinder Plus web application. Results Expression of a single NKG2D-L was sufficient to trigger NKX101 cytotoxicity against target cell lines. Flow cytometry analysis revealed that pre-treatment with Ara-C upregulated NKG2D-Ls on AML cell lines in a dose-dependent manner. NKX101 showed enhanced anti-leukemic activity against AML cells pre-treated with Ara-C compared to untreated controls, demonstrating that the combination can enhance NKX101 potency. Assessment of the combination of NKX101 and Ara-C using a dose-response matrix revealed that the two modalities combine to potently kill AML cells. Conclusions In summary, we show that NKX101 potently targets and eliminates AML cells expressing NKG2D-Ls further validating NKG2D-Ls as therapeutic targets. Additionally, we show that the combination of Ara-C and NKX101 leads to increased anti-leukemic activity, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML.

Tumor Treatment Response Assessed During the Chemo-Radiotherapy for Locally Advanced NSCLC

To evaluate the capability of assessing intratumoral treatment response distribution with using FDG-PET/CT during the chemoradiotherapy of locally advanced NSCLC. Twelve of total 50 patients with stage III NSCLC were enrolled in the institutional protocol for concurrent chemoradiotherapy with treatment dose of 54-60 Gy in 27-30 fractions. For each patient, a pre-treatment FDG-PET/CT image (SUV0) and a mid-treatment image (SUVm) obtained within the treatment dose of 24 ∼ 46 Gy were obtained. Followed by deformable PET/CT registration between SUV0 and SUVm, the tumor voxel SUV reduction ratio was obtained to construct a tumor dose response matrix (DRM). Tumor SUVavid was also constructed by limiting tumor voxel SUVm > a given value. Spatial correlations of the tumor SUV0, SUVm, SUVavid and DRM were determined. The mean and coefficient variation (CV) of the SUV0, SUVm and DRM for all tumors were 6.56(64%), 2.82(59%) and 0.52(70%) (Table contains the individual data), which were like those on the SUVs and the mean DRM of head-neck HPV- patients reported previously, but much larger on the DRM variation. The inter-tumoral CVs of SUV0 and DRM were 17% and 43%, which were much smaller than those of the intra-tumoral CVs 61% and 55%. Meanwhile, the intra-tumoral variations on both SUV0 and DRM were much larger than those of head-neck HPV- patients. There was a weak correlation between SUV0 and SUVm with the correlation coefficient 0.32, a medium correlation of -0.51 between SUV0 and DRM; 0.58 between SUVm and DRM. It implies that the rule of tumor dose response DRM on treatment modification decision cannot be fully replaced by either SUV0 or SUVm. The spatial correlation between tumor DRM and SUVavid was 0.23 with SUVavid value > 3, which was getting weaker when increasing SUVavid value. Spatial dose response for NSCLC assessed using FDG-PET/CT feedback demonstrated high treatment resistant patterns, which had a large intra-tumoral variation. In addition, the medium correlations of DRM vs SUV0 and DRM vs SUVm imply that all these factors could be used to guide adaptive modification of NSCLC treatment.

Tumor Treatment Response Assessed During the Concurrent Chemoradiotherapy for Nasopharyngeal Patients

To evaluate intratumoral treatment response distribution with using FDG-PET/CT during the chemoradiotherapy of nasopharyngeal patients (NPC). A total of 5 of 30 patients with stage III-IVA NPC were enrolled in the institutional protocol for induction/concurrent chemoradiotherapy with radiation dose of 70 Gy in 33 fractions. For each patient, a pre-radiation treatment FDG-PET/MRI image (SUV0) and a mid-treatment image (SUVm) at the treatment dose of 31.8 Gy were obtained. Followed by deformable PET/MRI registration between SUV0 and SUVm, the tumor voxel SUV reduction ratio was obtained to construct a tumor dose response matrix (DRM). Tumor SUVavid was also constructed by limiting tumor voxel SUVm > a given value. Spatial correlations of the tumor SUV0, SUVm, SUVavid and DRM were determined. The mean and coefficient variation (CV) of the SUV0, SUVm and DRM for all tumors were 5.05 (52%), 2.72 (49%) and 0.64 (63%) (Table contains the individual data), which were smaller than those on the SUVs of head-n-neck HPV+ patients reported previously due to the induction chemotherapy, but had much larger DRM mean and CV. The inter-tumoral CVs of SUV0 and DRM were 29% and 27%, which were much lower than those of the intra-tumoral CVs 43% and 57%. Meanwhile, the intra-tumoral variations on SUV0 was smaller than the one of head-neck HPV+ patients, but the DRM intra-variation was much larger. There was a weak correlation between SUV0 and SUVm with the correlation coefficient 0.13, a medium correlation of -0.55 between SUV0 and DRM, but a strong correlation, 0.72, between SUVm and DRM. However, the spatial correlation between tumor DRM and SUVavid was getting weaker as the SUVavid value increasing and equal 0.47 with SUVavid value > 3. The spatial dose response DRM for NPC in the concurrent chemoradiotherapy was relatively high, while had relatively low baseline tumor metabolic activity SUV0. It was most likely due to the induction chemotherapy. In addition, the tumor dose response showed vary large intra-tumoral variation. The high correlations between DRM and SUVm imply that SUVavid could be used partially to guide adaptive modification of NPC treatment with carefully selected boundary value.

Tumor Voxel Dose-Response Matrix Prediction Using Deep Learning

Tumor voxel dose-response matrix (DRM) can be assessed using a series of FDG-PET/CT feedback images acquired during radiotherapy. Predicting the tumor voxel DRM earlier is crucial for effectively implementing adaptive treatment management. However, it is also challenging due to FDG uptake dynamic fluctuation in tumor cells. This study investigated the feasibility of predicting tumor voxel DRM during the early treatment weeks using the advanced deep learning (DL) technique. Serial FDG-PET/CT images were acquired at the pretreatment (pre-Tx), the 2nd and 4th treatment weeks during standard chemo-radiotherapy (35 × 2 Gy) from each of the 50 patients with head and neck squamous cell carcinomas (HNSCC). The reference value of tumor voxel DRM (DRMref), representing the average metabolic change ratio during the treatment, was determined using a linear regression performed on the standard uptake values (SUV)s obtained at the pre-Tx (SUV0), the 2nd (SUV2) and the 4th (SUV4) treatment weeks following deformable PET/CT image registration. A DL model, 3D residual-Unet with a total of 3.4 million parameters, was trained to predict the tumor voxel DRMref with using the SUV0 and SUV2 matrices as inputs. The performance of the DL model was evaluated using 10-fold cross-validation and was compared to that of a linear regression (LR) model determined on the SUV0 and SUV2 matrices. The mean (SD) of the tumor voxel DRMref was 0.46 (0.2) over all 34612 tumor voxels. The predicted tumor voxel DRM was 0.5 (0.38) and 0.46 (0.15) for the LR model and the DL model, respectively. For those resistant voxels (23.7% of all tumor voxels) with a DRMref > 0.6, the DRM deviation was 0.13 (0.4) and -0.11 (0.13) for the LR model and the DL model, respectively. For those sensitive voxels (76.3%) with a DRMref ≤ 0.6, the DRM deviation was 0.01 (0.23) and 0.03 (0.08) for the LR model and the DL model, respectively. The proposed DL model can predict the tumor voxel DRM with a single FDG-PET feedback image acquired during the 2nd treatment week of radiotherapy for HNSCC patients. The prediction accuracy was improved compared to that of the LR model with a substantial reduction in the variances of the prediction errors. This work demonstrates the great potential of utilizing DL techniques to improve the efficiency of tumor response assessment and adaptive treatment management.

Adaptive Radiotherapy with Dose Fractionation Painting

Efficacy of dose fractionation is dependent on tumor radiosensitivity. However, due to intra-tumoral dose response heterogeneity, the advantage of fractionation cannot be fully utilized. FDG-PET/CT has been used to assess intra-tumoral dose response during the treatment course. Thus, radiosensitivity of different sub-volumes within the individual tumors can be assessed and used to design dose fractionation. For each patient, the pre-treatment FDG-PET/CT and a feedback FDG-PET/CT obtained within the 3rd week of the chemo-radiotherapy for HN cancer were used to construct tumor voxel dose response matrix, DRM. Biologically equivalent dose EQD2 was determined using the DRM and assuming relative stability of the radiation double-hit effect, meanwhile normal tissue EQD2 was determined assuming the corresponding α/β = 3.0. Tumor voxel EQD2 ratio was calculated with a given limitation of normal tissue EQD2 and used to determine tumor voxel fractionation (Table). Tumor was divided into few sub-volumes based on ranges of the tumor voxel DRM value. Different fractionation doses, but same number of fractions, were selected to ensure that the planning dose distribution can be simultaneously delivered. Table shows the EQD2 of tumor voxel DRM for each given fractionation and normal tissue dose limitation. The treatment dose efficiency increases exponentially as increasing dose per fraction for those of resistant tumor voxels, i.e., DRM > = 0.7. The 2 Gy per fraction was used within the first 3 treatment weeks before tumor voxel dose response assessed. After the 15 fractions, tumor voxel dose fractionation will be adjusted with respect to its DRM and the expected treatment dose. Typically, 3 sub-volumes for each resistant tumor were segmented and designed with 3 fractionation regimens. With respect to normal tissue EQD2 constraint < = 70 Gy, the corresponding EQD2 was 55 ∼ 73 Gy for tumor sub-volume with DRM = 0.1 ∼ 0.6; 77 ∼ 90 Gy for tumor sub-volume with DRM = 0.65 ∼ 0.75; > 101 Gy for tumor sub-volume with DRM > = 0.8 respectively. For very resistant tumor cells i.e., DRM > 0.9, the EQD2 > 160 Gy can be achieved or > 244 Gy if the normal tissue EQD2 constraint can be relaxed to 90 Gy for the small resistant tumor sub-volume. For the study patients, tumor sub-volume with DRM > 0.9 = 0.128 ∼ 7.4cc. Adaptive dose fractionation painting can be achieved followed by the dose response assessment. Dependent on the size and location of resistant tumor sub-volumes, tumor EQD2 could be largely improved without increasing normal tissue dose.

Abstract 3183: Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against solid tumors

Abstract Background: Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense against opsonized target cells wherein the Fc-fragment of an antibody binds to the CD16 Fc receptor (FcγRIIIa) on NK cells, triggering NK cell activation and targeted killing. Cetuximab is a human IgG1 antibody targeting epidermal growth factor receptor (EGFR) that is approved for the treatment of colorectal and head and neck cancers. Binding of cetuximab to EGRF+ cell lines has been shown to trigger ADCC mediated by NK cells. NKX101 is an investigational allogeneic NK cell therapy in development for the treatment of cancer. NKX101 is engineered to express an NKG2D-based chimeric antigen receptor (CAR) and membrane-bound IL-15. To investigate the combination of NKX101 and cetuximab, we developed in vitro assays to (i) demonstrate enhanced activity against cancer cells via CD16-mediated ADCC activity, (ii) compare ADCC activity of NKX101 cells generated from high-affinity CD16 and low-affinity CD16 donors, and (iii) evaluate the synergistic interaction between NKX101 and cetuximab. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. NucRed-labeled EGFR+ NKG2D-L+ human cancer cell lines were pre-incubated with cetuximab or isotype control and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte instrument. For synergy assessments, a dose response matrix of NKX101 cells and cetuximab was used to generate cytotoxicity values. Synergy scores for combinations were determined using the Synergy Finder Plus web application. To determine donor CD16 V/F 158 genotypes, genomic DNA was isolated from each donor and assayed using a TaqMan CD16 genotyping kit. Results: We demonstrate that cetuximab increases the anti-tumor effect of NKX101 in a dose-dependent manner. Assessment of the interaction between NKX101 and cetuximab in vitro revealed that the two agents can combine to kill cancer cells in a synergistic manner. Blocking CD16 on NKX101 cells with a neutralizing antibody significantly decreased potency of the combination, suggesting that the improvement in potency observed is a direct result of CD16-mediated ADCC activity. To determine if common CD16 polymorphisms influence NKX101 ADCC activity, we compared NKX101 cells generated from CD16 158 V/V, V/F, and F/F donors. No significant differences in potency were observed with NKX101 cells expressing the high affinity CD16 158 V/V genotype compared with those derived from CD16 lower affinity 158 V/F or 158 F/F donors. In summary, we show that the combination of an engineered allogeneic NK cell therapy, NKX101, with cetuximab leads to an increase in the specific killing of cancer cells in vitro, supporting further investigation into this combination for the treatment of solid tumors. Citation Format: Cynthia Cho, Kyle Hansen, Max Zhang, Carmel Chan, James Trager. Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3183.

In Vitro Evidence of Double Blockade of Asciminib with Reduced Dose of ATP-Binding Pocket Inhibitors in the Treatment of Chronic Myeloid Leukemia Harboring ABL1 Kinase Domain Mutation

Background Asciminib (ASC), a first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor, inhibiting BCR-ABL1 via allosteric binding. ASCEMBL trial showed superior efficacy of ASC to Bosutinib (BOS) with a higher molecular response rate, and a lower rate of discontinuation due to lack of efficacy (24.2% vs 35.5% at week 96). However, 24.2% of the patients still experienced lack of efficacy with ASC monotherapy. Also, out of the 39 patients who discontinued ASC due to lack of efficacy, 22 patients (56%) showed a new or persistent ABL1 kinase domain (KD) mutation either on ATP binding sites or Myristoyl pocket site. This finding suggests that ASC monotherapy is not always sufficient to overcome TKI-resistance particularly in those who already experienced multiple TKI failure. Accordingly, dual inhibition of ABL1 kinase protein with ASC combined with ATP binding pocket inhibiter (ABPI) is very attractive option to overcome TKI resistance. The remaining question is dose. A phase 1 study attempted to determine appropriate dose of ASC in combination with fixed dose of ABPIs including Imatinib (IMA), Dasatinib (DAS) and Nilotinib (NIL). But, this approach with fixed dose of ABPIs could add up toxicities not just from ABPIs but also from the combination of two drugs. Accordingly, a different strategy is to be developed to reduce the exposure to ABPI, while combining with ASC. In the present study, we simulated the combination treatment of ASC with ABPIs based on a clinically accessible fixed-dose for various ABL1 mutations to obtain answers to the following questions:Can combination of ASC with 4 different type of ABPIs be universally effective to suppress CML cell growth via dual blockade?Is there a specific ABPI for dual blockade in a specific ABL1 kinase domain mutation?What is the drug concentration of ABPIs for dual blockade when combined with fixed dose of ASC? Methods and Materials In order to simulate the combination treatment of ASC with ABPIs, the IC50 for ASC and ABPI measured from wild type K562 and BaF3BCR-ABL+cell lines were applied: ASC (3nM), IMA (150nM), BOS (10nM), DAS (0.8nM) and NIL (10nM) was applied in the wild type (WT) BaF3 cell lines and cell inhibition was assessed with WST-8 assay. 8 different BaF3 cell lines including wild type, G250E, Y253H, E255K, M244V, M351T, F317V and H369P mutant lines were treated with ABPI alone, ASC alone, and in combination by serial dilution based starting from the measured IC50 concentration, and the Dose-Response matrix was calculated and analyzed through SynergyFinder. Results A part of the results are summarized in the figure. As shown, with each ABPI at the baseline IC50 concentration in K562 cells, cell growth was inhibited to 59.8%, 56.9%, 61.5%, 59.9% and 71.6-79.4% with IMA, BOS, DAS, NIL and ASC (Fig A). When cell growth inhibition was assessed with combination treatment of reduced dose of ABPI and fixed dose of ASC (3nM), we observed that the combination treatment of ASC with ABPI can inhibit BaF3 cell growth even at a reduced concentration of ABPI from IC50 (Fig B). When combined of ABPI at baseline doses with 3nM ASC, 100% of cell inhibition was noted as expected. When treated with 3nM ASC with with 50% reduced dose of ABPI, 84.6-100% of cell inhibition was noted in WT, 99.2-99.6% in G250E, 96.8-99.4% in E255K mutant BaF3 cell lines, concluding that these cell lines were highly sensitive to double blockade; M351T and F317V BaF3 cell lines showed in the range between 30-77% of cell inhibition with a half dose of ABPI/baseline dose ASC, referring it intermediate sensitive cell lines; H369P, Y253F and M244V cell lines showed less effective cell inhibition compared to other cell lines, indicating the low sensitive cell lines. It implies that each cell line does have its own inherent profile of resistance to double blockade (Fig C). In addition, DAS and BOS seems to be better ABPIs for double blockade for intermediate to low sensitive mutations except the F317V mutation, which can be overcome with IMA or NIL combination. Conclusion The current study strongly suggests that 1) double blockade with reduced dose of ABPI in combination with fixed dose of ASC is effective to control CML cells with different ABL1 KD mutations. Each cell lines does have its inherent resistance level to double blockades, which needs to be further explored. A phase 1 study with reduced dose of ABPI in combination with fixed dose ASC is strongly warranted to define an optimal dose for combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dynamic Characteristics and Predictive Capability of Tumor Voxel Dose-Response Assessed Using 18F-FDG PET/CT Imaging Feedback.

PurposeTumor voxel dose–response matrix (DRM) can be quantified using feedback from serial FDG-PET/CT imaging acquired during radiotherapy. This study investigated the dynamic characteristics and the predictive capability of DRM.MethodsFDG-PET/CT images were acquired before and weekly during standard chemoradiotherapy with the treatment dose 2 Gy × 35 from 31 head and neck cancer patients. For each patient, deformable image registration was performed between the pretreatment/baseline PET/CT image and each weekly PET/CT image. Tumor voxel DRM was derived using linear regression on the logarithm of the weekly standard uptake value (SUV) ratios for each tumor voxel, such as SUV measured at a dose level normalized to the baseline SUV0. The dynamic characteristics were evaluated by comparing the DRMi estimated using a single feedback image acquired at the ith treatment week (i = 1, 2, 3, or 4) to the DRM estimated using the last feedback image for each patient. The predictive capability of the DRM estimated using 1 or 2 feedback images was evaluated using the receiver operating characteristic test with respect to the treatment outcome of tumor local–regional control or failure.ResultsThe mean ± SD of tumor voxel SUV measured at the pretreatment and the 1st, 2nd, 3rd, 4th, and last treatment weeks was 6.76 ± 3.69, 5.72 ± 3.43, 3.85 ± 2.22, 3.27 ± 2.25, 2.5 ± 1.79, and 2.23 ± 1.27, respectively. The deviations between the DRMi estimated using the single feedback image obtained at the ith week and the last feedback image were 0.86 ± 4.87, −0.06 ± 0.3, −0.09 ± 0.17, and −0.09 ± 0.12 for DRM1, DRM2, DRM3, and DRM4, respectively. The predictive capability of DRM3 and DRM4 was significant (p < 0.001). The area under the curve (AUC) was increased with the increase in treatment dose level. The DRMs constructed using the single feedback image achieved an AUC of 0.86~1. The AUC was slightly improved to 0.94~1 for the DRMs estimated using 2 feedback images.ConclusionTumor voxel metabolic activity measured using FDG-PET/CT fluctuated noticeably during the first 2 treatment weeks and obtained a stabilized reduction rate thereafter. Tumor voxel DRM constructed using a single FDG-PET/CT feedback image after the 2nd treatment week (>20 Gy) has a good predictive capability. The predictive capability improved continuously using a later feedback image and marginally improved when two feedback images were applied.

P344: THE DUAL BCL-2 AND BCL-XL INHIBITOR AZD4320 SHOWS ON-TARGET ACTIVITY IN ALL AND ACTS SYNERGISTICALLY WITH MCL-1 INHIBITION

Background: Targeting anti-apoptotic BCL-2 family proteins by BH3-mimetics has become a promising treatment strategy in acute lymphoblastic leukemia (ALL). Heterogeneous activity of the selective BCL-2 inhibitor venetoclax has been observed, but other anti-apoptotic proteins including BCL-XL promote venetoclax insensitivity. In clinical trials, targeting BCL-XL has previously resulted in a dose-limiting decrease of platelets. AZD4320 was developed as a dual inhibitor of BCL-2 and BCL-XL, and its dendrimer conjugate (AZD0466) was recently reported to demonstrate anti-tumor activity in hematological cancer models, while showing only a transient thrombocytopenia. Aims: In this study, the anti-leukemia activities of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of MCL-1-selective AZD5991 were evaluated and compared to the effects of other BH3-mimetics (BCL-2-selective venetoclax, BCL-XL-selective A-1331852 and MCL-1-selective S63845). The on-target activity of the inhibitors was functionally characterized and combination effects were analyzed. Methods: Cell viability assays were performed in ALL cell lines and patient-derived xenograft (PDX) samples analyzing half maximal effective concentrations (EC50). Protein complexes and expression of apoptosis regulators were analyzed by immunoprecipitation and immunoblotting. Dynamic BH3 profiling using synthetic BH3-peptides was performed to determine the dependency of ALL cells on BCL-2 family proteins. Combination effects were assessed by dose-response matrix analyses. Results: First, we determined the efficacy of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of the MCL-1 inhibitor AZD5991 for cell death induction in seven B-cell precursor (BCP) ALL cell lines and in a series of 13 PDX samples, showing heterogeneous responses. Interestingly, sensitivities of individual samples to both compounds were not associated with each other. However, we found a significant correlation of sensitivity to the dual inhibitor (AZD4320) with BCL-2 inhibition (venetoclax; N=13; rs=0.56; p=0.049), but no association with BCL-XL inhibition (A-1331852). Analyzing activities of all five BH3-mimetics including venetoclax, we found lowest EC50 values for AZD4320, indicating particular sensitivity for this dual inhibitor as compared to the single inhibitors (p<0.001). Investigating dependencies of ALL cells on BCL-2 family proteins by dynamic BH3 profiling, we found a shift towards MCL-1-dependence upon exposure to AZD4320, while AZD5991 induced an increased combined dependence on BCL-2 and BCL-XL, indicating potential synergistic activity of triple inhibition. Using co-immunoprecipitation analyses, we found that the exposure of ALL cells to AZD4320 reduced binding of both BCL-2 and BCL-XL to BIM, confirming on-target activity. Moreover, AZD5991 reduced binding of BIM to MCL-1. Accordingly, combined treatment with both inhibitors results in the release of BIM and downstream apoptosis signaling. Assessing combinatorial treatment in a primary PDX sample using multi-dose matrix analyses of both inhibitors revealed a positive mean Bliss synergy score of +4.8 indicating synergistic activity. Importantly, the highest synergism was found at low concentrations of both inhibitors, suggesting efficacy at moderate concentrations, which could potentially be achieved in vivo. Summary/Conclusion: In summary, our study demonstrates sensitivity, on-target activity and synergism of the dual BCL-2 and BCL-XL inhibitor AZD4320 with inhibition of MCL-1, thereby providing strong evidence for further clinical evaluation in ALL.

DDRE-28. IDENTIFICATION OF SYNERGISTIC DRUG COMBINATIONS FOR THE THERAPY OF IDHmut GLIOMA

Abstract OBJECTIVE Isocitrate Dehydrogenase (IDH) mutation in glioma results in a multitude of biological differences with consequences for survival and therapy response. Therefore, IDH mutated (IDHmut) and wildtype (IDHwt) tumors are regarded as separate entities with the need for adjusted therapy like the combination of procarbazine, CCNU and vincristine (PCV). However, as vincristine has often severe side effects like neuropathy new effective therapy options are required. Therefore, we searched for combinations of FDA-approved drugs which effectively inhibit the growth of IDHmut cells in vitro. METHODS We tested different drug combinations of a drug library consisting of 146 FDA-approved drugs on two established IDHmut GSC lines. Based on a previous single agent drug screen, six drugs were selected (Idarubicin, Ixazumib, Ponatinib, Neratinib, Romidepsin) to be combined with all 146 drugs of the library. Cell viability was assessed by the CellTiterGlo 3D assay (Promega) in 96 well plates, while Caspase-Glo 3/7 3D assay was used to measure induction of apoptosis. RESULTS Out of 1460 drug combinations tested altogether 21 synergistic drug combinations could be identified and validated. The combination with the highest blood-brain-barrier permeability score was further investigated. Finally, drug-concentrations elucidating the highest synergistic effect on proliferation was further studied in a 8-point dose-response matrix followed by validation in additional four IDHmut GSC lines. CONCLUSION This work can lay the foundation for future improvements of the therapy of patients suffering from LGGs.

Exploiting LY3009120 and Asciminib Combination to Target TKI-Resistant CML

The oncogenic BCR-ABL1 tyrosine kinase is the driver of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Tyrosine kinase inhibitors (TKIs) targeting ABL kinase are generally effective, but subsets of patients treated with single-agent TKIs develop resistance due to mutations in BCR-ABL1 that impair TKI binding. We have previously reported that BCR-ABL1 compound mutants (exhibiting two mutations within the same BCR-ABL molecule) that include the T315I gatekeeper mutation confer a high degree of resistance to all clinical ABL TKIs used as single agents, including ponatinib and the allosteric inhibitor asciminib. However, combining asciminib with ponatinib provides an effective strategy for overcoming compound mutation-based resistance (Eide et al. Cancer Cell 2019). As the clinical utility of ponatinib is limited by cardiovascular toxicity, including arterial occlusive events (AOEs), we decided to search for alternative molecules for use in combination with asciminib.To identify functional ponatinib analogs, we performed Quantum Similarity Modeling (QSM) on the reported crystal structure of T315I mutant ABL1 kinase in complex with nilotinib and asciminib (5MO4) (Wylie et al. Nature 2017) to search for other molecules. Compared to conventional computational modeling, QSM identifies novel classes of structurally distinct compounds that are comparable on a quantum level by precisely defining their interaction with the target. Affinity inferred by close complementarity with the shared ligand-protein surface in the region of the surveyed binding site is mapped, using multiple weak local associations. Our in silico QSM platform combines quantum methods with machine learning to investigate extensive chemical spaces. We screened several million compounds against BCR-ABL1 and identified 51 potential candidates predicted effectively to block T315I mutant BCR-ABL1 when combined with asciminib. To prioritize potent and non-toxic drug combinations for further development against compound mutants, we initially profiled all 51 compounds for their efficacy against Ba/F3 BCR-ABL T315I cells, alone and in combination with asciminib (1 nM). Of 51 compounds, LY3009120, a pan-RAF inhibitor that is currently in phase I clinical development for advanced solid malignancies (Sullivan et al. Mol Cancer Ther 2020), showed strong activity against BCR-ABL T315I when combined with asciminib. These data provided proof of principle for the QSM approach.We next tested the efficacy of all 51 candidates ± asciminib against Ba/F3 cells harboring T315I-inclusive BCR-ABL1 compound mutants, including Y253H/T315I, E255V/T315I, H396R/T315I, G250E/T315I, and T315L as the most resistant mutants. Neither single agent showed any effect. However, LY3009120 strongly inhibited BCR-ABL1 compound mutants when combined with asciminib. No toxicity was observed against Ba/F3 parental cells, confirming that the effects of the combinations are mediated by inhibition of BCR-ABL1. Synergy quantification of the dose-response matrix for the LY3009120/asciminib combination using the Zero Interaction Potency model demonstrated highly synergistic interactions (Synergy score > 10) between the two inhibitors. To directly assess the binding affinity of LY3009120 to the ABL1 kinase domain, we used the cell-based NanoBRET intracellular ABL1 kinase assay on HEK-293 cells expressing luciferase-tagged ABL1. The NanoBRET assay uses energy transfer to quantify the affinity of test compounds by competitive displacement of a cell-permeable fluorescent tracer that is reversibly bound to an ABL1-luciferase fusion protein. We found that LY3009120 competes off the fluorescent tracer at a low micromolar range (EC 50 = 0.75 μM), confirming direct binding of LY3009120 to the kinase domain of ABL1. We hypothesize that the binding of LY3009120 to the ABL1 kinase domain induces a conformational change that re-establishes asciminib binding to the myristoyl binding pocket, allowing for synergy. Studies to quantify the binding affinity of LY3009120 and asciminib to BCR-ABL1 mutants are underway, and data will be presented.In summary, our findings validate QSM as a novel in silico approach to identify TKI combinations. Combining LY3009120 with asciminib may be an effective, low-risk strategy to target BCR-ABL1 compound mutants in patients with clinical TKI resistance. DisclosuresDeininger: SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Novartis: Consultancy, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding.

Predictive Capability and Dynamic Characteristic of Tumor Voxel Dose-Response Assessed Using 18F-FDG PET/CT Imaging Feedback

To effectively implement FDG-PET/CT imaging feedback for tumor voxel treatment response assessment and treatment adaptation, this study investigated the predictive capability and the dynamic characteristic of tumor voxel treatment response with respect to the timing of imaging feedback.Serial FDG-PET/CT images were obtained at pretreatment and weekly during the standard chemoradiotherapy with treatment dose of 2Gy × 35 from 33 head and neck cancer patients. Reference of tumor voxel dose-response matrix (DRMref) was constructed for each patient using the average slope of tumor voxel PET intensity change ratio obtained from the first 4 weekly PET feedback images following deformable PET/CT image registration. The predictive capability of tumor voxel DRM assessed using 1 or 2 PET feedback images obtained during the first 4 treatment weeks was evaluated. The evaluation included the tumor voxel DRM correlation and the voxel DRM discrepancy between the predictions and the reference. The dynamic characteristics of DRM were studied by comparing the DRM assessed at different treatment weeks. The dynamic characteristics were also correlated to the tumor volume and tumor voxel location with respect to the human papillomavirus (HPV) status.The correlation coefficient between the DRMref and the DRMs predicted using a single feedback image increased from 0.82±0.2 for DRM2 at the 2nd treatment week to 0.95±0.03 for DRM4 at the 4th treatment week. The deviation between the tumor voxel DRMref and the predicted DRMs was (4.7±53.9)% for DRM2, (-7.3±21.1)% for DRM3, and (6.2±15.6)% for DRM4. The deviation reduced to (4.4±8.4)% or (1.1±7.8)% when using the week 2&4 or 3&4 feedback images. Tumor voxel improved its response significantly after 2nd treatment week in both HPV+ and HPV- tumors. In HPV- tumors, the magnitude of tumor voxel DRM reduction was correlated with the distance from the tumor surface to the voxels with a Spearman correlation coefficient = 1, P = 0.017.Tumor voxel treatment response showed large improvement after the 2nd treatment week with the delivered dose of 2Gy × 10 and got relatively stabilized after 2Gy × 15. FDG-PET/CT imaging acquired in the 3rd treatment week is recommended if single image feedback is selected. Feedback images obtained in the 2nd and 4th treatment weeks will additionally improve predictive capability and provide guidance for early treatment dose adjustment.