Single-agent Treatment Research Articles

Abstract A072: Strategies to improve KRAS G12D inhibitor (MRTX1133) therapy in pancreatic ductal adenocarcinoma

Abstract Background: More than 90% of pancreatic ductal adenocarcinomas (PDAC) harbor oncogenic KRAS, with KRAS G12D being the predominant mutation. Recently, a novel KRAS G12D inhibitor, MRTX-1133, has been developed, potentially revolutionizing PDAC treatment. However, clinical studies on KRAS G12C inhibitors suggest that there may be intrinsic resistance to KRAS inhibitors (KRASi) and acquired resistance can develop rapidly in patients. Moreover, our preliminary data suggest that acquired resistance mechanisms to chemotherapy can also confer partial resistance to KRASi. We hypothesize that effective killing of tumor cells with KRASi will require a rationally designed combinatorial treatment. Methods: We tested chemotherapy and targeted pathway inhibitors which can inhibit signaling upstream and downstream of KRAS to identify drugs that synergize with MRTX-1133. We treated resected human PDAC and mouse tumors from Kras-G12D; P53-R172H; PDX-Cre (KPC) prepared as organotypic slice cultures to test our identified drug combinations. We orthotopically inoculated KPC PDAC cells and treated mice with vehicle, single-agent drugs (MRTX1133 or Afatinib), or combination therapy. We established MRTX-1133-resistant (MRTXR) models in human SUIT2 cells, human organoids and mouse KPC cell lines to study the mechanisms of KRASi resistance. We performed RNA-seq and Reverse Phase Protein Array in these resistance models to identify pathways differentially activated during resistance development. Results: We found that irreversible ErbB inhibitors, Afatinib and Neratinib, had the greatest synergy with MRTX-1133 in parental and in MRTXR cells. We found that combination therapy with MRTX-1133 and Afatinib decreased the number of proliferating cells compared to the control or single-agent treatment in the human and mouse slice cultures. Combination therapy with MRTX1133 and Afatinib significantly reduced tumor growth in mice compared to the vehicle or single-agent controls. We also found that resistant cells demonstrated increased EGFR and HER2 phosphorylation compared to parental cells and that the MRTX-1133 and Afatinib drug combo synergistically decreased EGFR and HER2 phosphorylation. We identified and mapped the differentially expressed pathways and genes involved in KRASi resistance using RNA-seq and RPPA analyses. Conclusion: We identified a likely pathway of resistance to novel KRAS G12D inhibitors, and we demonstrate that this resistance can be effectively targeted using existing therapeutics targeting the ErbB pathway. Citation Format: Kevin Christian M. Gulay, Edgar Esparza, Xinlian Zhang, Evangeline S. Mose, Jonathan Weitz, Minya Pu, Karen Messer, Andrew M. Lowy, Herve Tiriac. Strategies to improve KRAS G12D inhibitor (MRTX1133) therapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A072.

Lyt-200, a Humanized Anti-Galectin-9 Antibody, Exhibits Preclinical Efficacy in Models of Hematological Malignancies

Patients with relapse and refractory (R/R) hematological malignancies typically have 5-year overall survival rates of less than 50% when receiving chimeric antigen receptor (CAR) T-cell therapy and less than 30% after receiving hematopoietic stem cell transplantation (HSCT). Human leukemia cells utilize a variety of biochemical mechanisms, which allow them to escape host immune surveillance. These molecular pathways cause impairment of the anti-cancer activities of immune cells, which could attack and kill leukemia cells. These dismal outcomes for patients with R/R disease highlight the need for novel treatment regimens when current therapeutic options are exhausted. Galectins are s-type lectins that promote diverse biological processes including adhesion, signaling, and immunosuppression. In acute myeloid leukemia (AML), galectin-9 (GAL-9) maintains the leukemia initiating cell (LIC) population and plays an important role in treatment resistance. We recently tested LYT-200, a fully humanized IgG4 αGAL-9 monoclonal antibody, developed by PureTech Health, which has so far been well tolerated with no dose limiting toxicities in a Phase I clinical trial for solid tumors (NCT04666688). We tested LYT-200 in vitro and in vivo across multiple hematological malignancies. Flow cytometry was used to assess the surface expression of GAL-9 on human B-ALL, AML, T-cell acute lymphoblastic leukemia (T-ALL), and diffuse large B-cell lymphoma (DLBCL) cell lines relative to receptor T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) and Programmed cell death protein-1 (PD-1) expression. Compared to healthy human peripheral blood mononuclear cells (PBMCs), where GAL-9 surface expression was low or absent depending on the immune cell, GAL-9 was highly expressed on the surface of all human blood cancer cell lines tested (>100-fold increase). Notably, GAL-9 expression was comparable to PD-1 surface expression and higher than that of TIM-3 on AML, B-ALL, T-ALL, and DLBCL cells. Treating all cell lines with LYT-200 relative to control immunoglobulin resulted in extensive cell death, which occurred after 48 and 72 hours of treatment. Furthermore, a side-by-side comparison with αTIM-3 antibody treatment at equivalent concentrations, revealed that LYT-200 treatment was significantly more effective at killing blood cancer cells at low doses of 10-100 ng/mL in vitro. The in vitro efficacy of LYT-200 against multiple blood cancer subtypes extended to in vivo protection and survival benefit in murine models of T-ALL and AML, both in immunocompromised and immunocompetent mice. When immunocompromised mice were transplanted with human T-ALL cell lines or patient-derived samples, single-agent treatment with LYT-200 (1.5 mg/kg/weekly) was comparable to survival results obtained with methotrexate (0.75 mg/kg/weekly) administration. Notably, the combination of LYT-200 and methotrexate treatments led to the best survival outcomes in both contexts. Similar results were obtained in xenograft studies of AML, where single-agent LYT-200 (1.5 mg/kg/weekly) treatment was more effective at protecting mice than cytarabine (100 mg/kg/weekly). However, combining both treatments resulted in the best survival outcomes with greater than 80% of mice surviving over 2 months after disease appearance. In all, our results demonstrate that GAL-9 is an important driver of multiple blood cancer subtypes and that targeting GAL-9 with LYT-200 represents a potential novel treatment strategy which warrants additional pre-clinical and clinical testing.

Asciminib As Initial Therapy for Patients with Chronic Myeloid Leukemia in Chronic Phase (CML-CP)-H. Jean Khoury Cure CML Consortium Study

Background: Although tyrosine kinase inhibitors (TKIs) have dramatically improved the outcome of patients with CML, there remains need for improvement as less than 50% achieve sustained MR4.5 by 10 years (sine qua non for treatment discontinuation) and approximately 40% need change of therapy within 5 years because of lack of efficacy or unacceptable toxicity. Asciminib is a potent allosteric inhibitor of BCR::ABL1, effective in vitro against BCR::ABL1 mutations that confer resistance to ABL-competitive tyrosine kinase inhibitors (TKIs). Asciminib has potential to combine with TKIs to prevent the emergence of resistant clones, potentially increasing the depth of molecular response in a greater number of patients compared with single-agent treatment. In a phase I study of asciminib, 48% of patients with CP-CML resistant to or intolerant of multiple prior TKIs achieved a major molecular response. The significant efficacy of asciminib in this setting suggests it might be more effective than other available options in achieving deep early responses in patients with newly diagnosed CML. Our goal in this study is to evaluate the role of asciminib in the treatment of patients with newly diagnosed CML-CP. Study Design and methods: This study is a multicenter phase 2, non-randomized open-label single-group study of asciminib in patients with newly diagnosed CP-CML (NCT05143840). The primary outcome is to evaluate the proportion of patients attaining MR4.5 within 12 months of therapy. In addition, patient reported outcomes will be collected at multiple timepoints. Patients will receive asciminib 40 mg orally twice daily. Peripheral blood BCR::ABL1 by RQ-PCR will be monitored in a central lab. Patients who do not achieved MR4.5 after 24 months of single agent asciminib will be offered the addition of nilotinib with the goal to attain MR4.5. In those instances, nilotinib will be started at 300 mg BID added to asciminib. Patients will discontinue study treatment if they experience disease progression, treatment failure according to ELN criteria or unacceptable toxicity. Patients who, after three years of asciminib +/- nilotinib, achieve sustained MR4.5 for at least 2 years may discontinue study treatment for an attempt at treatment-free remission. Sustained MR4.5 is defined as BCR::ABL1<0.0032% for approximately 2 years or more on at least 4 tests performed approximately 3 months apart. The primary objective of this study is to determine the rate of deep molecular response (DMR), defined as BCR::ABL1 <0.0032% IS by RQ-PCR at 12 months with asciminib compared to historical second generation TKI. With a total of 50 subjects, the study will have a 90% power to detect a 13.5% difference with an improvement of DMR at 12 months from 5% (based on historical experience with second generation TKIs used in frontline setting) to 18.5%. The exploratory endpoint of evaluating the rate of DMR after adding nilotinib at 24 months for patients not in DMR will be assessed at 36 months (after 12 months on combination therapy) and compared to the historical rate of 25% DMR with single agent nilotinib at 36 months. The study will have a 82% power to detect a 10% difference, 95% power to detect 15% difference and 99% power to detect a 20% difference.

Rituximab Infusion in 30 Minutes’ Is Safe and Improves the Flow of Outpatients with Lymphoma Treatment (SPEEDR)

Rituximab is a chimeric monoclonal antibody approved to treat B cell non-Hodgkin's lymphoma. Infusion reactions among patients are common during the first exposure but decrease with subsequent infusions. During the COVID-19 pandemic, there was an urgent need to avoid crowding of patients at our outpatient clinic at Karolinska University Hospital. We therefore introduced a new schedule for rapid IV infusion of 30 minutes of rituximab (SPEEDR) to patients who had well tolerated at least one dose of IV rituximab given in 90 minutes. We here present a retrospective analysis of tolerance and side effects for patients treated between January 2021 and December 2021. Ninety-nine patients received a total of 190 doses of rapid 30 minute infusion of rituximab. Fifty eight (59%) were male and the median age of our patients was 73 years. The patients had different types of lymphoid malignances and the most common diagnoses were diffuse large B-cell lymphoma (DLBCL, 38%), follicular lymphoma (FL, 21%) and marginal zone lymphoma (MZL, 11%). Thirty-four patients received single-agent rituximab treatment whereas 65 received rituximab in combination with chemotherapy. The 30 minute infusion was given after a preceding well-tolerated 90 minute dose. All doses thereafter were administrated in 30 minutes if no adverse events > grades 1 were seen. Rituximab was diluted in 500 ml 0.9% sodium chloride and infused in 30 minutes IV with a steady infusion rate without ramp up. The treating nurse measured blood pressure and pulse before starting the infusion and after completed infusion for all patients. Temperature was monitored before starting infusion and if the patient felt any discomfort during treatment or after. Data on infusion reactions, blood pressure, temperature and pulse were extracted from each patient's journal. Statistical analysis was performed using STATA. During the study period only 3 patients experienced an infusion reaction during or after the 30 minute infusion. No reaction was more than grade 2 and all patients responded well to standard treatment with fluids and could complete the rituximab infusion. Two of the patients who had an adverse event had not received any steroids before rituximab, one was treated with R-CHOP and had ingested 100 mg prednisone as part of the regimen. Analysis of median values before and during infusion for pulse was 70 vs 71.5 BPM (p=0.16), blood pressure 119/71 vs 119/70 (p= 0.08) mm Hg. Given the long serum half time of rituximab, the rapid SPEEDR infusion is highly unlikely to have negative effects on efficacy. We conclude that rituximab given as a 30-minute IV infusion is feasible and safe for patients with lymphomas who have previously tolerated a 90 minute infusion. This is an efficient way to optimize work flow in the outpatient clinic and to give cost-effective treatment to lymphoma patients.

The Menin Inhibitor Ziftomenib (KO-539) Synergizes with Agents Targeting Chromatin Regulation or Apoptosis and Sensitizes AML with MLL Rearrangement or NPM1 Mutation to Venetoclax

MLL1-rearranged (MLL-r) leukemias are dependent on the interaction of the adaptor protein menin with the oncogenic MLL (KMT2A) fusion protein. We previously demonstrated that NPM1 mutant (NPM1mut) acute myeloid leukemias (AMLs) exhibit a similar dependency on the menin interaction with wildtype MLL, a histone 3 lysine 4 methyltransferase. Pharmacologic inhibition of the menin-MLL interaction reverses MEIS1/HOX-driven leukemic gene expression, induces differentiation, and has anti-leukemic activity in preclinical NPM1mut and MLL-r AML models. Ziftomenib is currently assessed in clinical phase I/II trials and explorative efficacy against relapsed / refractory AML has been demonstrated. Here, we characterized the effects of ziftomenib on MLL-r and NPM1mut AML in detail and systematically screened for synergistic drug combination partners. First, we assessed the single-agent effects of ziftomenib on MLL-r (MOLM13, MV411, OCI-AML2) and NPM1mut (OCI-AML3) AML cell lines. Cellular assays demonstrated profound dose-dependent killing after seven days of treatment with IC50 values<25nM. HL60 and NB4 cells that are both wildtype for NPM1 and MLL served as negative controls and were not affected. Similar to previously characterized menin inhibitors (menin-i), ziftomenib uniformly induced transcriptional downregulation of MEIS1, PBX3, FLT3, and BCL2 in these leukemias, while genes upregulated upon ziftomenib were enriched for myelomonocytic differentiation processes. Ziftomenib-induced differentiation was also evidenced by cytology and upregulation of cell surface CD14 and CD11b. To identify synergistic combinations, we designed a screen assessing single and combined effects of ziftomenib and 37 targeted compounds (selected by confirmed preclinical or clinical activity against AML), each at four different concentrations and in a constant 1:4 ratio. We observed significant synergistic effects (combination index<0.5) of ziftomenib in MLL-r and NPM1mut AML, particularly pronounced for agents targeting chromatin regulation & DNA damage (e.g., LSD1, PRMT5, and PARP) as well as apoptosis & cell cycle (e.g. BCL2, MCL1, and CDK4/6). As we have demonstrated with other menin-i, ziftomenib induced transcriptional downregulation of FLT3 in the FLT3-mutant AML setting and also exhibited synergistic activity with the FLT3 inhibitor gilteritinib. As ziftomenib also uniformly downregulated BCL2, we assessed its combination with venetoclax in more detailed response assays and found synergistic growth inhibition compared to single treatment or vehicle control in all tested MLL-r and NPM1mut AMLs. While OCI-AML3 cells are not sensitive to venetoclax, the combination with ziftomenib was synergistic when using prolonged combined agent exposure (5 days) but not with standard 24-48h venetoclax treatment. shRNA-based knockdown of MEN1 (menin) also sensitized MOLM13 to venetoclax. Next, we found by Annexin V staining that the combination enhanced apoptosis compared to single agent treatment. Also, BCL2 homology (BH) 3 profiling revealed that ziftomenib treated MLL-r and NPM1mut AML cells exhibited a significantly enhanced cytochrome c release in response to BIM, PUMA, BAD, and MS1 peptides. These findings suggest that ziftomenib-induced apoptotic priming may contribute to synergistic effects with venetoclax in these leukemias. Next, we assessed the combination in a human MV411-derived leukemic xenograft model in vivo. We found significantly reduced leukemia burden defined by bone marrow engraftment after 14 days of treatment compared to the single agent or vehicle control animals. Single agent and combination assessment of four primary human NPM1mut AML patient samples in a human stroma cell co-culture model also showed the most pronounced anti-leukemic effects for the ziftomenib-venetoclax combination compared to all other controls. In summary, we demonstrated that ziftomenib has significant activity against MLL-r and NPM1mut AML and synergizes with certain agents targeting chromatin regulation and apoptosis. The combination of ziftomenib with venetoclax is particularly synergistic and already available for clinical trial assessment.

DHODH: A Promising New Therapeutic Target in Pediatric T-ALL

Acute T-cell lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in children and young adults. When compared to B-ALL, T-ALL has a higher rate of induction failure as well as relapse, with very poor overall survival in both scenarios. T-ALL has not benefitted from the same immune-mediated therapies (e.g., anti-CD19 CAR-T cells) and poses an ongoing treatment challenge as well as an unmet therapeutic need. Here, we demonstrate that inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme that converts dihydroorotate to orotate as part of the de novo synthesis of uridine, has a robust anti-leukemia effect in both in vitro and in vivo models of T-ALL. DHODH is ubiquitously-expressed, and inhibition of DHODH (DHODHi) leads to rapid depletion of pyrimidine ribo- and deoxyribonucleotides. A cell's ability to tolerate periods of pyrimidine starvation is dependent on a variety of alternative salvage pathways and is not well-understood. Clinical grade DHODH inhibitors are currently under investigation in trials assessing their effect in myeloid malignancies, where they are also known to have a pre-clinical anti-leukemic effect. We sought to understand the mechanism underlying the sensitivity of T-lymphoblasts to DHODHi, and to develop new DHODHi therapeutic combinations for patients with T-ALL. It is known that the intracellular concentration of nucleotides is high in activated T-lymphocytes, underlying the fact that two low-potency DHODHi's are approved for the treatment of autoimmune T-cell diseases (e.g., teriflunomide for multiple sclerosis and leflunomide for rheumatoid arthritis). We hypothesized that T-cells may have a particular addiction to de novo nucleotide synthesis and that this can be exploited as a vulnerability by the small molecule inhibition of DHODH. We demonstrated that T-ALL is highly sensitive to both chemical and genetic DHODHi using a variety of in vitro models (Figure A). Furthermore, we show that T-lymphoblasts respond to DHODHi by undergoing rapid cell cycle arrest and apoptosis. Additionally, we quantified the changes in intracellular nucleotides using PCA extraction and high-performance liquid chromatography (HPLC). The abundance of UTP, CTP, ATP, and GTP were determined across a panel of T-ALL cell lines at baseline and following treatment with a small molecular inhibitor of DHODH. The depletion of intracellular pyrimidines is rapid, falling to ~30% of baseline at 6 hours and confirming that the extracellular salvage is not able to meet the nucleotide demands of these cells even in the presence of complete media. Of note, the concentration of ATP and GTP are unaffected, confirming the specificity of DHODH on de novo pyrimidine synthesis as well as the viability of the cells over the 24-hour treatment. In several in vivo T-ALL models, we demonstrated that DHODH inhibition (specifically using the small molecule inhibitor brequinar) led to rapid, robust disease response (Figure B). These single agent treatment studies (brequinar, 50 mg/kg given every 3-days) were initiated between day 7 - 14 when the leukemias had engrafted and were established with detectable disease in the bone marrow. Three model systems were used: (1) a genetically engineered mouse model (GEMM) of immature T-ALL in which an activated intracellular NOTCH is expressed by retroviral transduction, (2) a transgenic mouse model of mature T-cell leukemia/lymphoma driven by the HTLV1 protein Tax, and (3) a human PDX from a young man with relapsed/refractory T-ALL. DHODH inhibitor therapy was effective in all three models, though clearly and most dramatically prolonged the survival in the GEMM model, driven by dysregulated NOTCH. We are now focused on elucidating the mechanisms by which T-lymphoblasts are so incredibly sensitive to inhibition of DHODH. New data suggests that changes in mitochondrial membrane potential and mitochondrial mass following exposure to DHODHi may offer additional insights into this sensitivity. We are also studying DHODHi in combination with other approved and experimental agents toward the goal of a new therapeutic combination that is safe, well-tolerated, and efficacious for this patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Oral Azacytidine in Patients with Relapsed/Refractory Angioimmunoblastic T-Cell Lymphoma: Final Analysis of the Oracle Phase III Study

Background: Angioimmunoblastic T-cell Lymphoma (AITL) and nodal lymphomas of follicular helper T-cell origin have in common frequent mutations affecting genes regulating DNA methylation and hydroxymethylation such as TET2, DNMT3A and IDH2. Preliminary studies suggested efficacy of the hypomethylating agent 5-azacytidine (Blood. 2018; 132(21):2305-09) in relapsed/refractory AITL patients. We present here the final results of the ORACLE study (NCT03593018), a phase III trial comparing CC-486, an oral form of 5-azacytidine, to single agent treatment chosen by the investigator. Patients & methods: Eighty-six patients with relapsed/refractory AITL or nodal follicular helper T-cell lymphoma were randomized between CC-486 (n=42) and investigator's choice (gemcitabine, n=24, bendamustine n=16, romidepsin n=4) between November 2018 and February 2021. CC-486 was given at a daily dose of 300 mg/day (200 mg/day in Asian patients, based on previous phase I pharmacokinetics results) every day for 14 days out of 28 day-cycles, until progression or unacceptable toxicity. Gemcitabine and bendamustine were both administered for 6 cycles, while romidepsin was administered until progression. Patients were evaluated for response using 2014 international working group CT-based criteria. The primary endpoint was progression-free survival (PFS) based on local assessment of progressive disease, and overall survival was a key secondary endpoint in this study. Main secondary endpoints were PFS based on central review, overall response rate (ORR), complete response (CR) rate and safety. Statistical hypotheses were built on a PFS of 12 months in the experimental arm vs 5 months in the standard arm (HR=0.417), with a one-sided alpha risk of 0.025 and 90% power. Results: Median age was 69 years (IQR 62-76), and the male/female ratio was 1.4. Patients had received a median of 2 (IQR 1-2) previous lines of treatment, 90.6% of them had advanced stage (III-IV), 57% had elevated LDH, 23.3% had an ECOG performance status >1, 31.4% had an IPI score 4-5 and 35.8% had a bone marrow involvement. Patients received a median number of 5.5 (IQR 2;14) cycles of CC486, and respectively 2 (1-4), 3.5 (2-6) and 13.5 (5-25) cycles of gemcitabine, bendamustine and romidepsin. The primary endpoint was analysed after a follow-up of 14.4 months. Median PFS in the CC-486 arm was 5.6 (95%CI, 2.66-8.11) months vs 2.8 (95%CI, 1.87-4.83) months in the standard arm (stratified log-rank test p=0.0421), with a hazard ratio of 0.634 (95%CI, 0.38; 1.07), which did not reach the required significance of p<0.025. Median overall survival was 18.4 months (95%CI, 12.9-31.5) in the CC-486 arm vs 10.3 (95%CI, 4.2-13.5) in the standard arm, with a hazard ratio of 0.557 (95%CI, 0.323-0.961). Best overall response and complete response rates at 3 months in the CC-486 arm were 33.3% (95%CI, 19.6%-49.5%) and 11.9% (95%CI, 4%-25.6%) respectively, vs 43.2% (95%CI, 28.3%-59.0%) and 22.7% (95%CI, 11.5%-37.8%) in the standard arm. The most frequent (>40%) treatment emergent adverse events (TEAEs) for the CC-486 versus (vs) standard arm respectively were: blood and lymphatic system disorders (76.2% vs 93%) [neutropenia (42.9% vs 58.1%) and thrombocytopenia (23.8% vs 48.8%)], infections (35.7% vs 67.4%), gastrointestinal disorders (71.4% vs 55.8%). At least one grade 3/4 TEAE occurred in 76.2% patients in CC-486 arm vs 97.7% in the standard arm, and at least one serious TEAE occurred in 26.2% patients in CC-486 arm vs 44.2% patients in the standard arm. Central pathological review confirmed the diagnosis of AITL and TFH PTCL in 69 and 9 cases. We detected TET2, RHOA, DNMT3A and IDH2 mutations in 68/77 (88%), 51/75 (68%), 24/75 (32%) and 25/75 (33%) samples. None of these mutations were associated with PFS or OS of the whole population or the 5-azacytidine treated patients. Conclusion: The trial did not meet its primary endpoint, most likely due to an optimistic hypothesis of PFS improvement, resulting in a study including 86 patients which could be underpowered to detect a clinically meaningful difference. 5-azacytidine had a favourable safety profile compared to standard of care and was associated with prolonged overall survival, suggesting that this drug might have a role in the treatment of TFH PTCL and could be investigated in combination with other agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PLX51107 Enhances the Anti-Tumor Activity of Targeted Therapy in Chronic Lymphocytic Leukemia When Combined with Venetoclax

Background: The development of drugs targeting BTK and BCL2 have dramatically improved the therapeutic landscape in chronic lymphocytic leukemia (CLL). However, resistance to these agents have been reported due to non-recurrent changes in oncogenic pathways and gene expression signatures. The bromodomain and extra terminal (BET) family proteins (BRD2, BRD3, BRD4 and BRDT) are epigenetic reader proteins that recognize acetylated lysine residues in histones. They play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Of these, the BRD4 protein is highly enriched in super enhancers and regulate gene transcription of relevant oncogenes such as MYC, CDK genes, cyclin-D1, BCL2 and MCL-1. BRD4 inhibitors (BRD4i) block the transcription of these key oncogenes through the displacement of BRDs and other epigenetic modifiers from chromatin. In this study, we investigated the preclinical activity of BRD4i PLX51107, and their cooperative role with BCL2 inhibitor Venetoclax in CLL. Methods: 56 primary CLL cells were isolated via histopaque-1077 (Sigma-Aldrich, St Louis MO) from peripheral blood apheresis samples from consenting adults using Roswell Park Comprehensive Cancer Center protocol CIC-01-16. Peripheral blood mononuclear cells (PBMC) were seeded at 3 x106/mL in a 384 or 6 well tissue culture plates and exposed to different doses of PLX51107 (1-20uM) as a single agent or in combination with Venetoclax (1-100nM) for 48 and 72hrs (Selleck Chemicals, Houston TX). Cell viability was assessed using Cell Titer-glo (Promega, Madison WI) with all data being normalized to untreated controls. Synergy was calculated using Calcusyn (Cambridge, UK) software to determine Coefficient of Synergy (CI) values. Anti-tumor effect was also analyzed by induction of apoptosis using Annexin-V/PI staining by flow cytometry at 72hrs. Cells pellets were saved for western blotting to determine expression of BRD4 members and different members of the B-cell receptor, NF-kB pathway as also the BCL-2 family of proteins. Bcl-2 family profiling was performed via Milliplex Bcl-2 family protein panels 1 and 2 to look at overall changes in Bcl-2 family expression. Results: Single agent exposure to PLX51107 displayed time and dose dependent induction of apoptosis of CLL cells (Figure 1a). Next, we examined the interaction between the PLX51107 and Venetoclax. In 72hr exposure the combination of PLX51107 with Venetoclax with an ATM deletion was synergistic in 4 of 8 samples and 4 of 7 samples with p53 deletion. Annexin/PI staining yielded apoptotic cell death in the single agent and combination exposure with Venetoclax. Western blotting showed a modulation of both BRD4 proteins as well as Bcl-2 family member proteins (Figure 1b). Bcl-2 family profiling via Milliplex panels demonstrated the ratio of Bcl-2/Bcl x/L was much higher in samples with a high Venetoclax IC50 (136/1) compared to Venetoclax low IC50 (54/1). (Figure 1) -A) Samples with p53 deletion and corresponding synergy. (B) BCL2 family protein expression of CLL sample 214c. Conclusions: Our data indicates that a) PLX51107 has an antitumor effect in CLL cells; b) the combinational treatment of Venetoclax with PLX51107 highly improved the apoptotic effect of single agent treatments. A better understanding of the key signaling pathways for CLL cells' proliferation and survival impacted by BRD4i in combination with Venetoclax would provide a proof-of-concept rationale for studies validating BRD4i as epigenetic approach to target BCR signaling in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Drug Combinations on AML Cell Lines' Treatment

Since the beginning of chemotherapy exist in medical world, few substantial therapeutic changes have been made for patients with acute myeloid leukemia (AML). In the last decade, remarkable improvements have been seen in treatment of AML with several new drugs receiving regulatory approval. Despite promising number of drugs and studies, the outcomes of patients with AML remain unsatisfactory. Even though these drugs passed the trial phases before clinical usage, there are still unexplained areas how drugs interact with each other on each patient who has different genetic profile and biological variations. Therefore, this study was designed for understanding how each drug works on each cell line, drug binary combinations show which synergy model (synergistic, antagonistic or additive), and if the models show a variation with same drug combination on different cell lines, how much strong is their combination effect. With this design, thirteen drugs and two targeted therapies were used in treatments of 5 cell lines. The results of single agent treatments of each cell line were used to determine the drug dosages and parameters of eight-by-eight binary combinations and eleven-by-eleven binary combinations with each drug. Daunorubicin, cytarabine, idarubicin, mitoxantrone, etoposide as elements of traditional AML treatment were consistent with their clinical reputation as they are part of the first line treatment. The rest of the drugs showed diverse range of response to treatment. Some of the drugs' single dose responses showed incomplete killing effect on AML cell lines but they still have impressive combination effect with some specific drugs. Some unusual treatment elements showed significant effect with specific combination on specific cell lines. An accepted treatment combination which is well known combination in clinic showed reverse effect on a cell line. A couple drug which are already in clinical usage before stem cell transplantation has an adverse interaction with its pair on a cell line. Both Loewe's Combination Index and Bliss over-excess synergy scores exhibited significant variability with regards to cell line. Clustering of drugs based on their synergy scores across all cell lines and drug combinations resulted in groups that segregated based on drug mechanism, yielding validity to our results. Additionally, we observed that some drugs behave very differently with regards to their synergistic potential from the rest of drugs in their class. A few drugs showed different type of behavior on different cell lines. Some combinations reflected strong synergism or antagonism. In conclusion, the results revealed multiple new potential synergistic drug pairs that could be very beneficial in the clinic as well as illuminate the necessity to account for the disease's heterogeneity not only when considering individual therapeutics but combination of them as well. And a few drugs showed strong antagonism on some combinations which are observed on only specific cell lines. Both synergistic and antagonistic responses are substantial candidates for new animal models and then clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Time-Limited Maintenance with Ibrutinib after First-Line Therapy and the Incremental Impact on Minimal Residual Disease (MRD) for Patients with Chronic Lymphocytic Leukemia (CLL)

Background: Inhibitors of Bruton's tyrosine kinase (BTKi) are approved and clinically used as indefinite treatments (until disease progression) irrespective of the depth of response in patients (pts) with CLL. Despite impressive, sustained efficacy, their ability (as single agent) to target residual disease clone post induction treatment in CLL is not well established. As indefinite treatment is associated not only with cumulative toxicity but also high cost and compromised quality of life, we investigated the benefit of time-limited maintenance treatment (3 years) with ibrutinib to assess the ability of a BTKi to further deepen antitumor response achieved post induction treatment. Methods: CLL pts (n=35) who had residual disease (by clinical or molecular assessment) after primary induction treatment were enrolled on a prospective phase 2 trial (NCT02649387). MRD assessed in blood (PB)/bone marrow (BM), was determined by 8-color flowcytometry (1x10-4) and defined as ≥1 CLL cell per 10,000 leukocytes or ≥0.01%. Patient were eligible if they had detectable residual (non-progressive) disease within 1 year of completing induction and no prior ibrutinib (or other BTKi) treatment. Ibrutinib 420mg PO QD was given on days 1-28 of a 28-day cycle for up to 36 cycles. Clinical (iwCLL 2008 response criteria) and molecule response MRD (on PB) was done at baseline and then every 3 cycles while on treatment. BM-MRD evaluation was conducted only if PB-MRD was negative (on two separate timepoints 3 months apart). Monitoring of the PB-MRD was continued for 24 months after completing time-limited maintenance with ibrutinib. Cumulative toxicity was assessed using the CTCAE v4.0 and iwCLL criteria. Results: Patient characteristics are summarized in Table 1. In 34 out of 35 (97%) patients, obinutuzumab alone was used as induction therapy. The median follow up is 38.6 months (range 0.9-73.2) and the median number of maintenance cycles completed is 34 (range 1-36). Of the 33 patients assessed for PB-MRD, 8 (24.2%) pts became MRD- on at one assessment. Five patients had BM-MRD assessment with 1 (2.8%) converting to MRD-. The median time to achievement of MRD- was 4.4 months (range 2.7-34.7). We noted that single agent ibrutinib was unable to deliver stringent MRD-negativity (defined as achievement of MRD- status both in PB and BM sustained over 2 consecutive evaluations at least 3 months apart).To assess if pre-maintenance response could impact MRD post-treatment; we note that out of the 8 pts that achieved MRD-, 2 (25%) started with CR and 6 (75%) with nPR/PR at registration. Deepening of response was noted in 5 pts (14.3%) after a median of 14 cycles [3 (8.6%) pts improved nPR to CR and 1(2.9%) went from nPR to CRi and 1 (2.9%) went from a CRi to a CR]. Nine patients proceeded to subsequent treatment with the median time to next treatment (mTTNT) of 32.5 m (range: 12.1 - 61.0). One patient died due to a failure to thrive and acute kidney injury on dialysis and six pts have progressed. The median PFS for the entire cohort was 41.1 m (95% CI: 39.8 - NE). The most commonly reported adverse events (AEs) (seen in >10% pts) regardless of attributions were thrombocytopenia (24 pts; 68.6%), anemia (12 pts; 34.3%), neutropenia (9 pts; 25.7%), lymphopenia (9 pts; 25.7%), hypertension (9 pts; 25.7%), leucopenia (6 pts; 17.1%), fatigue (5 pts; 14.3%), headache (4 pts; 11.4%), and skin infections (4 pts; 11.4%). Atrial fibrillation (AFib) was noted in 3 pts (8.6%). Conclusion: We conclude that BTK targeting with ibrutinib alone although able to clear PB-MRD (24.2%), had limited efficacy to eradicate BM-MRD (2.8%) demonstrating a differential impact in these two disease compartments. While the rate of complete MRD eradication was not necessarily high (when compared to recent data with BTKi/BCL2i combination regimens) MRD-kinetics over time demonstrated progressive decrease in disease burden and response improvement with maintenance treatment. The sequential single agent treatment approach was well tolerated, and the time-limited therapy aimed at minimizing cumulative toxicity seemed to be effective in lowering rate of Afib (compared to historical control ~16%). This data warrants further investigation of time-limited maintenance therapy with newer, more favorably tolerated BTKi and/or Bcl2i post induction treatment(s) to optimize response vs. cumulative toxicity. Further evaluation of sequential time-limited treatments in CLL is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Population Pharmacokinetics and Exposure-Response Analyses from Phase I-III Studies Support Copanlisib Dose Selection in Relapsed Indolent Non-Hodgkin Lymphoma

Introduction: Copanlisib is a novel PI3K inhibitor that is approved for single-agent treatment of relapsed follicular lymphoma in patients (pts) who have received ≥2 systemic therapies. In the pivotal Phase III CHRONOS-3 study, pts with relapsed iNHL were randomized to copanlisib at the approved maximum tolerated dosing regimen of 60 mg administered on days 1, 8, and 15 of a 28-day cycle in combination with standard rituximab (C+R) or placebo plus rituximab (P+R) and demonstrated superior progression-free survival (PFS) of C+R vs P+R (hazard ratio [HR]: 0.52; Matasar et al. Lancet Oncol 2021). In an updated 1-year follow-up analysis, the benefit of C+R was confirmed in the overall relapsed iNHL population (Zinzani et al. EHA 2022). No dedicated dose-finding studies have been undertaken to investigate copanlisib dose selection when used in combination with rituximab; however, initial safety assessments demonstrated acceptable tolerability. We investigated popPK and ER relationships for copanlisib efficacy and safety from a pooled analysis, with particular focus on the 1-year follow-up of CHRONOS-3 to confirm copanlisib dose selection. Methods: A copanlisib comprehensive popPK model was developed from 712 pts across 9 copanlisib clinical Phase I-III studies. Demographic, laboratory, and co-medication covariates were investigated for their influence on copanlisib between-patient PK variability. Individual static and time-varying exposure estimates for pts enrolled in CHRONOS-3 were derived to investigate relationships to efficacy (PFS) and key treatment-emergent safety events using Cox proportional hazards (CPH) and multivariate logistic regression analyses after accounting for predefined potentially prognostic demographic, laboratory, and/or disease-related baseline covariates. Results: Copanlisib PK was best described by a 3-compartment PK model with first-order elimination following intravenous infusion. Copanlisib PK was dose proportional and time independent, with no accumulation with the approved dosing regimen. Statistically selected covariates influencing copanlisib PK included rifampin (strong CYP3A inducer) and itraconazole (strong CYP3A inhibitor) co-administration and mild or worse hepatic impairment, confirming the results of dedicated clinical pharmacology studies. Additional covariates influencing copanlisib PK included female sex, residence in Japan, and a CHRONOS-3 study effect, with the majority of covariates or subpopulations defined by sex, age, body weight, renal function, and geographic region showing exposure variations not exceeding traditional bioequivalence ranges (80-125%). In CHRONOS-3, CPH analyses demonstrated a statistically significant, positive ER relationship for PFS for all 3 time-varying copanlisib exposure estimates (average concentration [Cavg] over 2 weeks [Cavg2wk]: p=0.002; 4 weeks [Cavg4wk]: p=0.004; and 8 weeks [Cavg8wk]: p=0.002), along with a near-significant relationship for area under the curve from 0 to 168 hours (AUC(0-168)nd) [p=0.023] (Figure 1). Therefore, greater copanlisib exposure was associated with prolonged PFS, while lower copanlisib doses may result in reduced efficacy in relapsed iNHL. CPH and/or logistic regression analyses demonstrated no significant ER relationship for investigated safety events (serious adverse events [AEs], grade ≥3 treatment-emergent AEs, hyperglycemia, hypertension, diarrhea, nausea, fatigue, lung infections, and neutropenia of any grade). Thus, lower starting doses of copanlisib are not anticipated to improve safety and tolerability. ER analyses support the overall positive benefit/risk assessment of copanlisib reported from the clinical efficacy and results of CHRONOS-3 in iNHL. Conclusion: PopPK and ER analyses substantiate administration of a dosing regimen of copanlisib 60 mg on days 1, 8, and 15 of a 28-day cycle in the relapsed iNHL population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity invitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. Invivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.

Whole transcriptome sequencing analysis of synergistic combinations of plant-based antimicrobials and zinc oxide nanoparticles against Campylobacter jejuni

The emergence of antibiotic resistance among animal farms impels the development of novel antimicrobials or strategies for agri-food production. The combinational use of agents to achieve a synergistic antimicrobial effect provides many advantages such as dosage reduction, shortened treatment time, and avoidance of antimicrobial resistance. In this study, we evaluated the killing efficacy of single agent or combinational use of three antimicrobials, including cinnamon oil, encapsulated curcumin and zinc oxide nanoparticles (ZnO NPs), against a leading foodborne pathogen Campylobacter jejuni. We then investigated the antimicrobial mechanism using whole transcriptome sequencing analysis (RNA-Seq). The single-agent treatment of cinnamon oil, encapsulated curcumin, or ZnO NPs showed a significant antimicrobial effect against C. jejuni by generating more than 8-log reduction within 3 h. The transcriptional signatures of C. jejuni in response to these agents varied, indicating that these agents shared distinct mechanisms of action and were likely to generate synergistic effects. Cinnamon oil affected the integrity of cell membrane, which might lead to an increase in cell permeability. Encapsulated curcumin and ZnO NPs disrupted bacterial outer membranes and cell membranes against the same membrane protein targets. The combinational use of these agents showed synergistic antimicrobial effects and distinct mechanisms of action compared to single treatment. The combination of cinnamon oil and encapsulated curcumin provoked the expression of cellular signaling, but repressed the chemotaxis-associated genes. The antimicrobial resistance associated genes showed a low expression level in the combination of encapsulated curcumin and ZnO NPs. The tri-combination treatment systematically overexpressed genes involved in the amino acid synthesis, protein translation, and membrane protein synthesis. This study provides new insights in combating Campylobacter with minimizing the development of antimicrobial resistance in long-term usage.

Targeting depletion of myeloid-derived suppressor cells potentiates PD-L1 blockade efficacy in gastric and colon cancers

ABSTRACT Myeloid-derived suppressor cells (MDSCs) have been demonstrated to suppress antitumor immunity and induce resistance to PD-1/PD-L1 blockade immunotherapy in gastric and colon cancer patients. Herein, we found that MDSCs accumulate in mice bearing syngeneic gastric cancer and colon cancer. Death receptor 5 (DR5), a receptor of TNF-related apoptosis-inducing ligand (TRAIL), was highly expressed on MDSCs and cancer cells; targeting DR5 using agonistic anti-DR5 antibody (MD5-1) specifically depleted MDSCs and induced enrichment of CD8+ T lymphocytes in tumors and exhibited stronger tumor inhibition efficacy in immune-competent mice than in T-cell-deficient nude mice. Importantly, the combination of MD5-1 and anti-PD-L1 antibody showed synergistic antitumor effects in gastric and colon tumor-bearing mice, resulting in significantly suppressed tumor growth and extended mice survival, whereas single-agent treatment had limited effect. Moreover, the combination therapy induced sustained memory immunity in mice that exhibited complete tumor regression. The enhanced antitumor effect was associated with increased intratumoral CD8+ T-cell infiltration and activation, and a more vigorous tumor-inhibiting microenvironment. In summary, our findings highlight the therapeutic potential of combining PD-L1 blockade therapy with agonistic anti-DR5 antibody that targets MDSCs in gastric and colon cancers.

Exploiting endogenous and therapy-induced apoptotic vulnerabilities in immunoglobulin light chain amyloidosis with BH3 mimetics

Immunoglobulin light chain (AL) amyloidosis is an incurable hematologic disorder typically characterized by the production of amyloidogenic light chains by clonal plasma cells. These light chains misfold and aggregate in healthy tissues as amyloid fibrils, leading to life-threatening multi-organ dysfunction. Here we show that the clonal plasma cells in AL amyloidosis are highly primed to undergo apoptosis and dependent on pro-survival proteins MCL-1 and BCL-2. Notably, this MCL-1 dependency is indirectly targeted by the proteasome inhibitor bortezomib, currently the standard of care for this disease and the related plasma cell disorder multiple myeloma, due to upregulation of pro-apoptotic Noxa and its inhibitory binding to MCL-1. BCL-2 inhibitors sensitize clonal plasma cells to multiple front-line therapies including bortezomib, dexamethasone and lenalidomide. Strikingly, in mice bearing AL amyloidosis cell line xenografts, single agent treatment with the BCL-2 inhibitor ABT-199 (venetoclax) produces deeper remissions than bortezomib and triples median survival. Mass spectrometry-based proteomic analysis reveals rewiring of signaling pathways regulating apoptosis, proliferation and mitochondrial metabolism between isogenic AL amyloidosis and multiple myeloma cells that divergently alter their sensitivity to therapies. These findings provide a roadmap for the use of BH3 mimetics to exploit endogenous and induced apoptotic vulnerabilities in AL amyloidosis.

AML-256 A Phase 1 Study of Gilteritinib in Combination With Induction and Consolidation Chemotherapy in Patients With Newly Diagnosed Acute Myeloid Leukemia: Final Study Results

Gilteritinib, an oral FLT3 inhibitor, demonstrated antileukemic responses in patients with FLT3mut+ relapsed/refractory AML. Present final data from a phase 1 study of once-daily gilteritinib plus intensive chemotherapy in newly diagnosed (ND) AML. This 4-part, open-label study (NCT02236013) assessed gilteritinib dose/schedule and effects of alternative anthracycline treatment. Gilteritinib plus 7+3 induction with idarubicin or daunorubicin, plus ≤3 cycles of high-dose cytarabine and gilteritinib consolidation, and 2 years of single-agent gilteritinib maintenance treatment post consolidation or transplant. Adults with ND AML; FLT3 mutation not required for enrollment (known core binding factor fusions excluded). Safety, tolerability, PK, antileukemic effects. Eighty patients were allocated to gilteritinib (safety analysis set, n=78 [age 23-77 y]; FLT3mut+, n=44 [FLT3-ITD, n=33]). Median follow-up was 37.7 months. On-study HSCT was performed in 27/80 (33.8%) patients (FLT3mut+, 26/44 [59.1%]). Dose-limiting toxicities occurred in 15/78 (19.2%) patients (200-mg/d cohort: neutropenia, n=1; neutropenic colitis, n=1). Maximum tolerated dose was 120 mg/d. Across the study, AEs led to gilteritinib discontinuation in 24.4% of patients. At end-of-treatment, composite complete remission (CRc: complete remission [CR] + CR with incomplete hematologic recovery [CRi] + CR with incomplete platelet recovery [CRp]), CR, CRi, and CRp rates were 79.7%, 54.4%, 21.5%, and 3.8%, respectively, in the overall population and 90.9%, 70.5%, 13.6%, and 6.8% among FLT3mut+ patients. In the overall (n=79) and FLT3mut+ populations (n=44), 60-day mortality rates were 1.3% and 0%, respectively, and median (95% CI) disease-free survival was 15.1 (9.5, 31.9) and 15.1 (4.9, 31.9) months. In the overall (n=69) and FLT3mut+ populations (n=41) receiving ≥80 mg/d, median (95% CI) OS was 38.6 (21.7, NE) and 45.9 (30.8, NE) months, respectively, and the estimated 3-year OS rate was 56.0% and 60.1%. Anthracycline choice did not substantially impact CRc rate or toxicity. In patients who achieved CRc with gilteritinib ≥120 mg/d, mutational clearance of FLT3-ITD (summed FLT3 ITD:wildtype signal ratio ≤10-4) after consolidation was 84.6% (11/13). Gilteritinib plus induction and consolidation chemotherapy was well tolerated and resulted in CR, FLT3-ITD clearance, and long-term survival in patients with ND FLT3mut+ AML, supporting ongoing randomized trials testing this approach against current standards.

Extracts of Rheum palmatum and Aloe vera Show Beneficial Properties for the Synergistic Improvement of Oral Wound Healing.

Various local and systemic factors compromise oral wound healing and may lead to wound dehiscence, inflammation, or ulcers. Currently, there is a lack of topical therapeutical options. Thus, this study aimed to investigate the effect of Aloe vera (AV) and Rheum palmatum root (RPR) on oral wound healing capacity in vitro. The effect of AV and RPR on human primary fibroblast viability and migration was studied by measuring metabolic activity and gap closure in a scratch assay. Furthermore, cell cycle distribution and cytoskeletal features were analyzed. Antimicrobial activity against the oral pathogen Porphyromonas gingivalis was evaluated by broth microdilution assay. AV and RPR increased fibroblast migration after single agent treatment. Synergistic effects of the plant extract combination were observed regarding cellular migration which were confirmed by calculation of the phenomenological combination index (pCI), whereas the cell cycle distribution was not influenced. Furthermore, the combination of AV and RPR showed synergistic antibacterial effects as determined by the fractional inhibitory concentration index. This study demonstrated that the combination of AV and RPR can promote the migration of human primary fibroblasts in vitro and exert antimicrobial efficacy against P. gingivalis, suggesting these compounds for the topical treatment of wound healing disorders.

Preclinical assessment of synergistic efficacy of MELK and CDK inhibitors in adrenocortical cancer

BackgroundAdrenocortical cancer (ACC) is a rare and aggressive cancer with dismal 5-year survival due to a lack of effective treatments. We aimed to identify a new effective combination of drugs and investigated their synergistic efficacy in ACC preclinical models.MethodsA quantitative high-throughput drug screening of 4,991 compounds was performed on two ACC cell lines, SW13 and NCI-H295R, based on antiproliferative effect and caspase-3/7 activity. The top candidate drugs were pairwise combined to identify the most potent combinations. The synergistic efficacy of the selected inhibitors was tested on tumorigenic phenotypes, such as cell proliferation, migration, invasion, spheroid formation, and clonogenicity, with appropriate mechanistic validation by cell cycle and apoptotic assays and protein expression of the involved molecules. We tested the efficacy of the drug combination in mice with luciferase-tagged human ACC xenografts. To study the mRNA expression of target molecules in ACC and their clinical correlations, we analyzed the Gene Expression Omnibus and The Cancer Genome Atlas.ResultsWe chose the maternal embryonic leucine zipper kinase (MELK) inhibitor (OTS167) and cyclin-dependent kinase (CDK) inhibitor (RGB-286638) because of their potent synergy from the pairwise drug combination matrices derived from the top 30 single drugs. Multiple publicly available databases demonstrated overexpression of MELK, CDK1/2, and partnering cyclins mRNA in ACC, which were independently associated with mortality and other adverse clinical features. The drug combination demonstrated a synergistic antiproliferative effect on ACC cells. Compared to the single-agent treatment groups, the combination treatment increased G2/M arrest, caspase-dependent apoptosis, reduced cyclins A2, B1, B2, and E2 expression, and decreased cell migration and invasion with reduced vimentin. Moreover, the combination effectively decreased Foxhead Box M1, Axin2, glycogen synthase kinase 3-beta, and β-catenin. A reduction in p-stathmin from the combination treatment destabilized microtubule assembly by tubulin depolymerization. The drug combination treatment in mice with human ACC xenografts resulted in a significantly lower tumor burden than those treated with single-agents and vehicle control groups.ConclusionsOur preclinical study revealed a novel synergistic combination of OTS167 and RGB-286638 in ACC that effectively targets multiple molecules associated with ACC aggressiveness. A phase Ib/II clinical trial in patients with advanced ACC is therefore warranted.

Current Molecular Combination Therapies Used for the Treatment of Breast Cancer.

Breast cancer is the second leading cause of death for women worldwide. While monotherapy (single agent) treatments have been used for many years, they are not always effective, and many patients relapse after initial treatment. Moreover, in some patients the response to therapy becomes weaker, or resistance to monotherapy develops over time. This is especially problematic for metastatic breast cancer or triple-negative breast cancer. Recently, combination therapies (in which two or more drugs are used to target two or more pathways) have emerged as promising new treatment options. Combination therapies are often more effective than monotherapies and demonstrate lower levels of toxicity during long-term treatment. In this review, we provide a comprehensive overview of current combination therapies, including molecular-targeted therapy, hormone therapy, immunotherapy, and chemotherapy. We also describe the molecular basis of breast cancer and the various treatment options for different breast cancer subtypes. While combination therapies are promising, we also discuss some of the challenges. Despite these challenges, the use of innovative combination therapy holds great promise compared with traditional monotherapies. In addition, the use of multidisciplinary technologies (such as nanotechnology and computer technology) has the potential to optimize combination therapies even further.

Abstract A018: YAP signaling promotes resistance to MEK and AKT inhibition in NF1-related MPNSTs

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are a rare and deadly sarcoma with few therapeutic options that are the leading cause of death for patients with Neurofibromatosis Type 1 (NF1). MPNSTs are characterized by a large burden of genomic alterations, chemoresistance, and a 5-year survival rate of 25-50%. NF1 is the major negative regulator of RAS, therefore a hallmark of MPNSTs is deregulated RAS/MAPK signaling. To date, no targeted therapies have been approved for MPNST treatment, highlighting the need for an understanding of adaptive signaling mechanisms that drive resistance. The HIPPO pathway is a key regulator of organ growth and cellular differentiation and is a central driver of resistance to RAS pathway inhibition in other cancers. The key HIPPO effector, YAP1, interacts with RAS and AKT signaling pathways, creating alternate routes for resistance in multiple cancers. In our studies, we identified strong YAP activation in response to MEK and AKT inhibition in MPNST cell lines. Since YAP acts as a transcriptional co-activator through interaction with TEAD transcription factors, we proposed that targeting the Hippo pathway in MPNST will abrogate MAPK inhibitor resistance. To investigate tumor signaling responses and efficacy in vivo, we utilized 3 genomically distinct MPNST patient-derived xenograft (PDX) models. To assess drivers of MPNST resistance, we have developed a preclinical model of drug resistance that simulates clinical treatment schedules. Using a cross-over and a drug holiday design, we are able to evaluate patterns of response and resistance to resumed treatment. We observed distinctive responses to MEK and AKT inhibitors in each PDX line; however, the impact on MPNST growth was minimal with single agent treatment. Comparison of pathway activation between the “drug holiday” biopsies and the treatment endpoint, demonstrated distinct YAP and RAS (pERK) activation levels in resistant tumors. For example, strong YAP activation is observed in correlation with decreased pERK in resistant tumors. YAP activation was strongest at the invasive edge of viable tumor regions. Currently, we are evaluating the efficacy and signaling adaptations to investigate the underlying molecular mechanisms in these MPNST models. Overall, we have demonstrated the development of a clinically relevant model of MPNST resistance and a potential switch between RAS and YAP signaling that promotes resistance to MEK or AKT inhibition. Citation Format: Lauren McGee, Curt Essenburg, Lisa Turner, Angela Hirbe, Anwesha Dey, Jason Zbieg, Carrie Graveel, Matt Steensma. YAP signaling promotes resistance to MEK and AKT inhibition in NF1-related MPNSTs [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A018.