Single-agent Treatment Research Articles

DNMT and EZH2 inhibitors synergize to activate therapeutic targets in hepatocellular carcinoma

The development of more effective targeted therapies for hepatocellular carcinoma (HCC) patients due to its aggressiveness is urgently needed. DNA methyltransferase inhibitors (DNMTis) represented the first clinical breakthrough to target aberrant cancer epigenomes. However, their clinical efficacies are still limited, in part due to an “epigenetic switch” in which a large group of genes that are demethylated by DNMTi treatment remain silenced by polycomb repressive complex 2 (PRC2) occupancy. EZH2 is the member of PRC2 that catalyzes the placement of H3K27me3 marks. EZH2 overexpression is correlated with poor HCC patient survival. We tested the combination of a DNMTi (5-aza-2′-deoxycytidine, DAC) and the EZH2 inhibitor (EZH2i) GSK126 in human HCC cell lines on drug sensitivity, DNA methylation, nucleosome accessibility, and gene expression profiles. Compared with single agent treatments, all HCC cell lines studied showed increased sensitivity after receiving both drugs concomitant with prolonged anti-proliferative changes and sustained reactivation of nascently-silenced genes. The increased number of up-regulated genes after combination treatment correlated with prolonged anti-proliferation effects and increased nucleosome accessibility. Combination treatments also activate demethylated promoters that are repressed by PRC2 occupancy. Furthermore, 13–31% of genes down-regulated by DNA methylation in primary HCC tumors were reactivated through this combination treatment scheme in vitro. Finally, the combination treatment also exacerbates anti-tumor immune responses, while most of these genes were downregulated in over 50% of primary HCC tumors. We have linked the anti-tumor effects of DAC and GSK126 combination treatments to detailed epigenetic alterations in HCC cells, identified potential therapeutic targets and provided a rationale for treatment efficacy for HCC patients.

Abstract A20: Treatment of elderly patients with Peripheral T-Cell Lymphoma (PTCL): A single institution experience

Abstract Introduction: While standard treatment for younger patients with PTCL is chemotherapy and HD consolidation with stem cell rescue, treatment of o lder patients with PTCL is less defined, due to lack of clinical trial data and comorbidities due to ageing. Here we detail our single center outcomes of patients who were 70 years of age or older. Methods: We retrospectively reviewed the clinical characteristics, treatment used and outcomes of patients 70 years of age or older who were treated at Roswell Park Cancer Center for PTCL between 1/1/2001 to 12/31/2021. The patients were divided into three groups, group 1 received single agent therapy, group 2 received multiagent chemotherapy and group 3 received palliative radiation or no therapy. The baseline characteristics were described using descriptive statistics and survival were compared using Kaplan Meier method. Results: Forty elderly T cell lymphoma patients were treated at our center. The median age of whole cohort was 77 years, with 24 males and 16 females. Thirty patients had stage 3/4 disease at the time of diagnosis. The most common diagnosis were ALK -ve ALCL (n=14) and AITL (n=14), while ten patients had PTCL-NOS, and one patient had ALK +ve ALCL and PTCL-TFH each. Seven patients received single agent treatment (G1, n=7), of which six received brentuximab while one received lenalidomide. Twenty nine patients received multiagent treatment (G2, n=29), of which 22 patients received CHOP/CHOEP based therapy, 4 reveived HyperCVAD, and 3 received other chemotherapy treatments. Four patients received no treatment or palliative radiation only (G3,n=4). The patients in G1 were older (median age 85 years), compared to G2 (median age 74 years) and G3 (median age 75 years). The median ECOG of G1, G2 and G3 were 2, 0 and 1 respectively. The ORR of patients in G1 and G2 was 6/7 and 18/29 with CR of 4/7 and 14/24 respectively. Three patients in G2 died due to complications related to induction chemotherapy. Only two patients received autologous transplant after multiagent therapy, none of patients in G1 received an autologous transplant. The median overall survival of patients in G1, G2, and G3 was 25 months, 31 months and 2 months respectively, with no statistically significant difference between G1 and G2 (p=0.63). The median progression free survival of G1 and G2 was 27 months and 14 months (p=0.7). Conclusions: Older patients with T-cell lymphoma can be treated with personalized treatment based on comorbidities and performance status. We found that single agent treatment in selected group of patients produced statistically similar PFS and OS as multiagent treatment. Citation Format: Muhammad Salman Faisal, Gabrielle Hartman, Francisco Hernandez-Ilizaliturri, Sucharita Sundaram, Pallawi Torka, Paola Ghione. Treatment of elderly patients with Peripheral T-Cell Lymphoma (PTCL): A single institution experience [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A20.

Definition of a new blood cell count score for early survival prediction for non-small cell lung cancer patients treated with atezolizumab: Integrated analysis of four multicenter clinical trials.

ImportanceBlood cell count test (BCT) is a robust method that provides direct quantification of various types of immune cells to reveal the immune landscape to predict atezolizumab treatment outcomes for clinicians to decide the next phase of treatment.ObjectiveThis study aims to define a new BCTscore model to predict atezolizumab treatment benefits in non-small lung cell cancer (NSCLC) patients.Design, Setting, and ParticipantsThis study analyzed four international, multicenter clinical trials (OAK, BIRCH, POPLAR, and FIR trials) to conduct post-hoc analyses of NSCLC patients undergoing atezolizumab (anti–PD-L1) single-agent treatment (n = 1,479) or docetaxel single-agent treatment (n = 707). BCT was conducted at three time points: pre-treatment (T1), the first day of treatment cycle 3 (T2), and first day of treatment cycle 5 (T3). Univariate and multivariate Cox regression analyses were conducted to identify early BCT biomarkers to predict atezolizumab treatment outcomes in NSCLC patients.Main Outcomes and MeasuresOverall survival (OS) was used as the primary end point, whereas progression-free survival (PFS) according to Response Evaluation Criteria in Solid Tumors (RECIST), clinical benefit (CB), and objective response rate (ORR) were used as secondary end points.ResultsThe BCT biomarkers of neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) at time point T3 and neutrophil-to-monocyte ratio (NMR) at time point T2 with absolute cutoff values of NLR_T3 = 5, PLR_T3 = 180, and NMR_T2 = 6 were identified as strong predictive biomarkers for atezolizumab (Ate)–treated NSCLC patients in comparison with docetaxel (Dtx)–treated patients regarding OS (BCTscore low risk: HR Ate vs. Dtx = 1.54 (95% CI: 1.04–2.27), P = 0.031; high risk: HR Ate vs. Dtx = 0.84 (95% CI: 0.62–1.12), P = 0.235). The identified BCTscore model showed better OS AUC in the OAK (AUC12month = 0.696), BIRCH (AUC12month = 0.672) and POPLAR+FIR studies (AUC12month = 0.727) than that of each of the three single BCT biomarkers.Conclusion and RelevanceThe BCTscore model is a valid predictive and prognostic biomarker for early survival prediction in atezolizumab-treated NSCLC patients.

1041P Efficacy and safety of chemotherapy after progression on immunotherapy: Results of a multicenter retrospective observational trial

Limited evidence is to date available about the efficacy and safety of chemotherapy after progression on previous immunotherapy in metastatic NSCLC patients. We retrospectively collected data on consecutive, stage IIIB-IV, ECOG performance status (PS) 0-2, NSCLC patients treated with single agent or combination chemotherapy after progression on a previous immunotherapy regimen. Baseline characteristics, outcome measures and toxicities were recorded. An exploratory analysis on the predictive value of the neutrophil-to-lymphocyte (N/L) ratio was performed. One-hundred patients were included in the analysis. Median age was 67 (range 39-81) years, M/F 66%/34%, ECOG PS 0/1/≥2 47%/51%/2%, adeno/squamous carcinoma 77%/23%, with median of 50% (range 0-100) PD-L1 expression. Previous immunotherapy consisted on a single agent treatment in 83% of cases with a prevalence of pembrolizumab use with a median pre-chemotherapy N/L ratio of 4. Overall median time-to-progression on previous immunotherapy was 6 months while it raised to 7.5 months in cases receiving single agent. Only 33% of cases received chemotherapy after immunotherapy. Platinum doublets (mostly carboplatin) were delivered in 31% while single agent chemotherapy in 69% of cases (vinorelbine 25%, taxanes 25%, gemcitabine 8%) with a median of 4 (range 1-16) cycles delivered. Overall response rate was 21% with a median clinical benefit of 55%. Median time to progression was 4 (range 1-17) months and median overall survival was 5 (range 1-22) months. Comparison of low vs high N/L ratio subgroups did not show any significant difference in terms of survival. A minority of advanced NSCLC patients received chemotherapy after immunotherapy. Chemotherapy showed modest clinical efficacy after progression on immunotherapy while no safety issues were recorded. Baseline N/L ratio failed to predict chemotherapy benefit after immunotherapy.

1553O Health-related quality of life (HRQoL) in the phase III TROPiCS-02 trial of sacituzumab govitecan (SG) vs chemotherapy in HR+/HER2- metastatic breast cancer (MBC)

The antibody-drug conjugate SG comprises an anti-Trop-2 antibody coupled to SN-38 via a hydrolyzable linker. In the ongoing randomized TROPiCS-02 trial, SG is being evaluated vs single-agent chemotherapy treatment of physician’s choice (TPC) after CDK 4/6 inhibitor, endocrine therapy, and at least 2 chemotherapies in HR+/HER2- MBC setting. Here we assess the impact of SG on HRQoL in TROPiCS-02. Patients (pts) with locally determined HR+/HER2- MBC following (2-4 prior chemotherapies) for metastatic disease, were randomized to receive SG (10 mg/kg IV on days 1 and 8 of a 21-day treatment [Tx] cycle) or TPC (capecitabine, eribulin, gemcitabine, or vinorelbine). HRQoL was assessed at baseline (BL), day 1 of each Tx cycle (CxDx) and at end of treatment visit using the EORTC QLQ-C30. The analysis included all pts with valid HRQoL assessments at BL and ≥1 post-BL visit. Linear mixed-effect models for repeated measures were used to estimate differences in overall least-square mean changes from BL between Tx arms, using on-Tx data up to C11D1 (n ≥25 in both arms). The analysis included 446 pts (236 SG, 210 TPC; median age 56 y). The EORTC QLQ-C30 completion rate was ≥85% up to C13D1 in both Tx arms. Mean HRQoL scores at BL were generally similar for SG and TPC. The SG arm showed a trend of improvement in most HRQoL domains. A significantly greater improvement in physical functioning and dyspnea was observed for SG vs TPC and greater worsening in diarrhea (table).Table: 1553OEORTC QLQ-C30 least-square mean change from baselineSG vs TPC (95% CI)Global health status/QoL2.4 (-0.7, 5.5)FunctioningPhysical3.9 (0.9, 6.9)Role3.1 (-1.2, 7.4)Emotional0.9 (-2.4, 4.3)Cognitive-0.2 (-3.3, 2.9)Social0.8 (-3.2, 4.7)Summary Score0.6 (-1.8, 3.1)SymptomsFatigue-3.3 (-7.1, 0.5)Pain-1.6 (-5.5, 2.3)Nausea/Vomiting1.4 (-0.9, 3.8)Dyspnea-4.3 (-8.5, -0.1)Insomnia-3.7 (-8.1, 0.8)Appetite Loss-3.3 (-7.3, 0.8)Constipation-1.0 (-5.5, 3.4)Diarrhea10.4 (6.3, 14.5)Financial Difficulties0.9 (-2.5, 4.2)Positive value represents improvement for functioning and worsening for symptoms.SG, sacituzumab govitecan; TPC, treatment of physician’s choice. Open table in a new tab SG, sacituzumab govitecan; TPC, treatment of physician’s choice. Despite the association with worsening diarrhea, SG demonstrated an overall HRQoL benefit in heavily-pretreated patients with HR+/HER2- MBC.

Safety analyses from the phase 3 ASCENT trial of sacituzumab govitecan in metastatic triple-negative breast cancer

Sacituzumab govitecan (SG) is an anti-Trop-2 antibody-drug conjugate with an SN-38 payload. In the ASCENT study, patients with metastatic triple-negative breast cancer (mTNBC) relapsed/refractory to ≥2 prior chemotherapy regimens (≥1 in the metastatic setting), received SG or single-agent treatment of physician’s choice (eribulin, vinorelbine, capecitabine, or gemcitabine). This ASCENT safety analysis includes the impact of age and UGT1A1 polymorphisms, which hinder SN-38 detoxification. SG demonstrated a manageable safety profile in patients with mTNBC, including those ≥65 years; neutropenia/diarrhea are key adverse events (AE). Patients with UGT1A1 *28/*28 genotype versus those with 1/*28 and *1/*1 genotypes had higher rates of grade ≥3 SG-related neutropenia (59% vs 47% and 53%), febrile neutropenia (18% vs 5% and 3%), anemia (15% vs 6% and 4%), and diarrhea (15% vs 9% and 10%), respectively. Individuals with UGT1A1 *28/*28 genotype should be monitored closely; active monitoring and routine AE management allow optimal therapeutic exposure of SG.

Chk1 inhibition-induced BRCAness synergizes with olaparib in p53-deficient cancer cells

ABSTRACT Although targeting DNA-damage repair by inhibition of PARP exhibits weak or modest single-agent activity due to the existence of functional BRCA1/2 alleles, PARP inhibitors have been gradually applicable in BRCA-proficient cancers. Checkpoint kinase 1 (Chk1) inhibition selectively disrupts homologous recombination (HR)-mediated DNA repair and confers synthetic lethality in p53-deficient tumors, we therefore aim at expounding the chemopotentiating effects of Chk1 inhibition on PARPi in BRCA-proficient and p53-deficient cancer cells. Initially, BRCA wild-type, p53-null cells including AsPC-1 and H1299 demonstrated innate resistance to PARP inhibitor olaparib compared to BRCA1-mutant, p53-null MDA-MB-436 cells. We quantified the interaction between olaparib and a selective Chk1 inhibitor MK-8776, which produced synergistic effects under sub-IC50 concentrations in p53-depleted AsPC-1 and H1299 cells. Olaparib in combination with MK-8776 showed enhanced antitumor effects through prohibiting proliferation and secondarily inducing apoptosis in two cell lines. Of note, we observed that MK-8776 significantly sensitized cells to olaparib by broad DNA and chromosomal breaks. Mechanistically, MK-8776 abrogated olaparib-induced BRCA1 intranuclear foci formation, MCM7-mediated replication machineries, and ultimately triggered an accumulation of γH2AX, a well-recognized marker of DNA double-strand breaks. Additionally, we established ectopic expression of hotspot mutant p53 in H1299 cells. Introduction of p53R175 H promoted olaparib resistance as single-agent treatment, but the synergy between olaparib and MK-8776 was still achievable and the region of synergy was produced by lower combination concentrations. These data provide insight into how Chk1 inhibition could be effectively targeted and confer sensitivity to olaparib toward p53-deficient and HR-proficient cancers.

Stress-Mediated Responses of Aedes aegypti (Diptera: Culicidae) Larvae When Exposed to Metarhizium brunneum (Hypocreales: Clavicipitaceae) and Toxorhynchites brevipalpis (Diptera: Culicidae)

Aedes aegypti mosquitoes are capable of vectoring a wide range of diseases including dengue, yellow fever, and Zika viruses, with approximately half of the worlds’ population at risk from such diseases. Development of combined predator–parasite treatments for the control of larvae consistently demonstrates increased efficacy over single-agent treatments, however, the mechanism behind the interaction remains unknown. Treatments using the natural predator Toxorhynchites brevipalpis and the entomopathogenic fungus Metarhizium brunneum were applied in the laboratory against Ae. aegypti larvae as both individual and combined treatments to determine the levels of interaction between control strategies. Parallel experiments involved the removal of larvae from test arenas at set intervals during the course of the trial to record whole body caspase and phenoloxidase activities. This was measured via luminometric assay to measure larval stress factors underlying the interactions. Combined Metarhizium and Toxorhynchites treatments were seen to drastically reduce lethal times as compared to individual treatments. This was accompanied by increased phenoloxidase and caspase activities in combination treatments after 18 h (p < 0.001). The sharp increases in caspase and phenoloxidase activities suggest that combined treatments act to increase stress factor responses in the larvae that result in rapid mortality above that of either control agent individually. This work concludes that the underlying mechanism for increased lethality in combined parasite–predator treatments may be related to additive stress factors induced within the target host larvae.

Effective Combination Immunotherapy with Oncolytic Adenovirus and Anti-PD-1 for Treatment of Human and Murine Ovarian Cancers.

Simple SummaryThis study was conducted to find a new, more efficient, treatment for ovarian cancer. A combination of an oncolytic adenovirus (TILT-123) with immune checkpoint inhibitors was employed to treat ex vivo patient samples and was found statistically significantly more effective than control treatments ex vivo and showed potent efficacy towards in vivo tumor growth.Ovarian cancer (OvCa) is one of the most common gynecological cancers and has the highest mortality in this category. Tumors are often detected late, and unfortunately over 70% of OvCa patients experience relapse after first-line treatments. OvCa has shown low response rates to immune checkpoint inhibitor (ICI) treatments, thus leaving room for improvement. We have shown that oncolytic adenoviral therapy with Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (aka. TILT-123) is promising for single-agent treatment of cancer, but also for sensitizing tumors for T-cell dependent immunotherapy approaches, such as ICI treatments. Therefore, this study set out to determine the effect of inhibition of the immune checkpoint inhibitors (ICI), in the context of TILT-123 therapy of OvCa. We show that simultaneous treatment of patient derived samples with TILT-123 and ICIs anti-PD-1 or anti-PD-L1 efficiently reduced overall viability. The combinations induced T cell activation, T cells expressed activation markers more often, and the treatment caused positive microenvironment changes, measured by flow cytometric assays. Furthermore, in an immunocompetent in vivo C57BL/6NHsda mouse model, tumor growth was hindered, when treated with TILT-123, ICI or both. Taken together, this study provides a rationale for using TILT-123 virotherapy in combination with TILT-123 and immune checkpoint inhibitors together in an ovarian cancer OvCa clinical trial.

Final overall survival results from SOLO3: Phase III trial assessing olaparib monotherapy versus non-platinum chemotherapy in heavily pretreated patients with germline BRCA1 - and/or BRCA2-mutated platinum-sensitive relapsed ovarian cancer (026)

<b>Objectives:</b> In the open-label Phase III SOLO3 trial (NCT02282020), olaparib monotherapy provided clinically relevant and statistically significant improvements in objective response rate (ORR; primary endpoint) and progression-free survival (PFS; secondary endpoint), compared with single-agent non-platinum chemotherapy, in patients (pts) with germline <i>BRCA1</i> and/or <i>BRCA2</i>-mutated (g<i>BRCA</i>m) platinum-sensitive relapsed ovarian cancer (PSROC) who had received ≥2 prior lines of platinum-based chemotherapy (Penson <i>et al. JCO</i> 2020). We report final overall survival (OS) and second disease progression results from SOLO3. <b>Methods:</b> Pts were randomized (2:1) to olaparib tablets (300 mg bid) or single-agent non-platinum chemotherapy treatment of physician's choice (TPC; paclitaxel [P], topotecan [T], gemcitabine [G], or pegylated liposomal doxorubicin [PLD]). Study treatment continued until objective radiological disease progression, unacceptable toxicity, or other discontinuation criteria were met. The time from randomization to second progression or death (PFS2) and OS were secondary endpoints. As prespecified, this analysis was performed at approximately 60% data maturity for OS. <b>Results:</b> 266 pts were randomized (olaparib, <i>n</i>=178; TPC, <i>n</i>=88 [PLD, <i>n</i>=47; P, <i>n</i>=20; G, <i>n</i>=13; T, <i>n</i>=8]); 12 (14%) TPC pts withdrew before receiving study treatment. At the final data cut-off (DCO; April 16, 2021), 19 (11%) olaparib pts versus no TPC pts were still receiving study treatment. The percentage of pts who left the study prior to death was approximately 2.3 times higher in the TPC arm (22 pts [25%]) than in the olaparib arm (19 pts [11%]). Following disease progression, the majority of pts received subsequent anticancer therapy (119 of 178 [67%] olaparib pts vs 54 of 88 [61%] TPC pts received ≥1 subsequent anticancer regimen); three of 178 (2%) olaparib pts versus 23 of 88 (26%) TPC pts received a PARP inhibitor in their first subsequent line of therapy, with nine of 178 (5%) versus 33 of 88 (38%) pts, respectively, receiving a PARP inhibitor in any subsequent line of therapy. At the final DCO, PFS2 favored olaparib over TPC, although the between-group difference was not statistically significant, and OS was similar in the olaparib and TPC groups (see Table). Adverse events (AEs) were consistent with the known safety profile of olaparib and with previous SOLO3 analyses; no new safety signals were identified. Discontinuation of study treatment due to AEs occurred in 18 of 178 (10%) olaparib pts versus 15 of 76 (20%) TPC pts in the safety analysis set. Circulating tumor DNA analyses are ongoing. <b>Conclusions:</b> In the primary analysis of SOLO3, olaparib monotherapy improved ORR and PFS compared with single-agent non-platinum chemotherapy in heavily pretreated pts with g<i>BRCA</i>m PSROC. In the final analysis, PFS2 favored olaparib monotherapy over TPC, and OS was similar in both treatment groups, supporting the use of olaparib as a chemotherapy-free treatment option in this pt population. No new safety signals were identified.

Inhibition of mutant IDH1 promotes cycling of acute myeloid leukemia stem cells.

Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy invivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients.

Bioinformatics Analysis Reveals FOXM1/BUB1B Signaling Pathway as a Key Target of Neosetophomone B in Human Leukemic Cells: A Gene Network-Based Microarray Analysis.

Abnormal expression of Forkhead box protein M1 (FOXM1) and serine/threonine kinase Budding uninhibited by benzimidazoles 1 (BUB1B) contributes to the development and progression of several cancers, including chronic myelogenous leukemia (CML). However, the molecular mechanism of the FOXM1/BUB1B regulatory network and the role of Neosetophomone-B (NSP-B) in leukemia remains unclear. NSP-B, a meroterpenoid fungal secondary metabolite, possesses anticancer potential in human leukemic cells lines; however, the underlying mechanism has not been elucidated. The present study aimed to explore the role of NSP-B on FOXM1/BUB1B signaling and the underlying molecular mechanism of apoptosis induction in leukemic cells. We performed gene expression profiling of NSP-B-treated and untreated leukemic cells to search for differentially expressed genes (DEGs). Interestingly BUB1B was found to be significantly downregulated (logFC -2.60, adjusted p = 0.001) in the treated cell line with the highest connectivity score among cancer genes. Analysis of TCGA data revealed overexpression of BUB1B compared to normal in most cancers and overexpression was associated with poor prognosis. BUB1B also showed a highly significant positive correlation with FOXM1 in all the TCGA cancer types. We used human leukemic cell lines (K562 and U937) as an in vitro study model to validate our findings. We found that NSP-B treatment of leukemic cells suppressed the expression of FOXM1 and BUB1B in a dose-dependent manner. In addition, NSP-B also resulted in the downregulation of FOXM1-regulated genes such as Aurora kinase A, Aurora kinase B, CDK4, and CDK6. Suppression of FOXM1 either by siRNA or NSP-B reduced BUB1B expression and enhanced cell survival inhibition and induction of apoptosis. Interestingly combination treatment of thiostrepton and NSP-B suppressed of cell viability and inducted apoptosis in leukemic cells via enhancing the activation of caspase-3 and caspase-8 compared with single-agent treatment. These results demonstrate the important role of the FOXM1/BUB1B pathway in leukemia and thus a potential therapeutic target.

The combination of decitabine and aspirin inhibits tumor growth and metastasis in non-small cell lung cancer.

ObjectiveCombination therapy has become the hallmark of lung cancer treatment, as it reduces the dosage intensity of individual drugs while increasing their efficacy. In the current study, we analyzed the combinatorial effect of decitabine and aspirin on non-small cell lung cancer (NSCLC) cell growth.MethodsIn this study, we investigated the combinatorial effect of decitabine and aspirin by MTT, colony formation, and Transwell assays. We also explored the underlying molecular mechanism via a series of in vitro and in vivo experiments.ResultsThe combination of decitabine and aspirin regulated cell viability and migration in vitro. Moreover, the combination therapy suppressed tumor cell growth by inhibiting the β-catenin/STAT3 signaling pathway. Our study also found that the regimen increased the phosphorylation of β-catenin and decreased the expression of STAT3 and β-catenin.ConclusionThe combined administration of decitabine and aspirin significantly reduced tumor growth compared with single-agent treatment and the control in vivo. The study results indicated that decitabine and aspirin could suppress NSCLC cell growth and metastasis via the β-catenin/STAT3 signaling pathway.

Hymeglusin Enhances the Pro-Apoptotic Effects of Venetoclax in Acute Myeloid Leukemia

Venetoclax is used for the priority treatment of elderly patients with acute myeloid leukemia (AML). Resistance or intolerance to venetoclax offsets its clinical benefits in some patients. Combination strategies with other drugs are promising alternatives to overcome the current complications associated with venetoclax use. Hymeglusin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMGCS1), regulates the mevalonate pathway, which is vital for AML growth and chemosensitivity. The effects of the combination of venetoclax and hymeglusin on AML were explored in this study. The correlations between HMGCS1 and apoptosis-related genes were analyzed using the Gene Expression Profiling Interactive Analysis 2 and The Cancer Genome Atlas databases. Apoptosis and cell viability were detected in HL-60 and KG-1 cells after treatment with gradient concentrations of venetoclax or hymeglusin. The transcriptomic profiles of HL-60 and KG-1 cells were compared via RNA-Seq analysis. The effects of venetoclax and hymeglusin on apoptosis were validated in primary cells. The results showed that HMGCS1 expression was closely associated with apoptosis-related genes based on the data from large clinical databases. B cell lymphoma (BCL)-2 expression was elevated in AML and negatively associated with overall survival. Hymeglusin decreased BCL2 expression levels in HL-60 and KG-1 cells. Venetoclax and hymeglusin inhibited cell viability in both cell lines, but induced apoptosis in HL-60 cells. This discrepancy in sensitivity to hymeglusin may be attributed to the positive increase in the expression levels of HMGCS1 and multiple upregulated pro-leukemia genes in KG-1 cells. Combination treatment with venetoclax and hymeglusin significantly increased the apoptotic rates compared to single-agent treatment in both AML cell lines and primary AML cells. Furthermore, the combination strategy did not result in remarkably enhanced toxicity in normal mononuclear cells. Collectively, hymeglusin enhanced the effects of venetoclax on apoptosis. This combination strategy showed enhanced antileukemic activity with acceptable toxicity in AML.

Clinical Presentation, Treatment Outcomes, and Resistance-related Factors in South American Women with Low-risk Postmolar Gestational Trophoblastic Neoplasia.

There are few multinational studies on gestational trophoblastic neoplasia (GTN) treatment outcomes in South America. The purpose of this study was to assess the clinical presentation, treatment outcomes, and factors associated with chemoresistance in low-risk postmolar GTN treated with first-line single-agent chemotherapy in three South American centers. Multicentric, historical cohort study including women with International Federation of Gynecology and Obstetrics (FIGO)-staged low-risk postmolar GTN attending centers in Argentina, Brazil, and Colombia between 1990 and 2014. Data were obtained on patient characteristics, disease presentation, and treatment response. Logistic regression was used to assess the relationship between clinical factors and resistance to first-line single-agent treatment. A multivariate analysis of the clinical factors significant in univariate analysis was performed. A total of 163 women with low-risk GTN were included in the analysis. The overall rate of complete response to first-line chemotherapy was 80% (130/163). The rates of complete response to methotrexate or actinomycin-D as first-line treatment, and actinomycin-D as second-line treatment postmethotrexate failure were 79% (125/157), 83% (⅚), and 70% (23/33), respectively. Switching to second-line treatment due to chemoresistance occurred in 20.2% of cases (33/163). The multivariate analysis demonstrated that patients with a 5 to 6 FIGO risk score were 4.2-fold more likely to develop resistance to first-line single-agent treatment (p = 0.019). 1) At presentation, most women showed clinical characteristics favorable to a good outcome, 2) the overall rate of sustained complete remission after first-line single-agent treatment was comparable to that observed in developed countries, 3) a FIGO risk score of 5 or 6 is associated with development of resistance to first-line single-agent chemotherapy.

α-Ketoglutarate-Mediated DNA Demethylation Sustains T-Acute Lymphoblastic Leukemia upon TCA Cycle Targeting.

Simple SummaryA promising anti-cancer strategy is to target the tumor’s dependence on particular nutrients. However, resistance to single-agent treatment is common due to the adaptability of cancer cells. This research seeks to understand how T acute lymphoblastic leukemia (T-ALL) escapes disruption of the tricarboxylic acid (TCA) cycle, a biochemical pathway critical for T-ALL survival. We show that leukemic cells modify their DNA to increase the activity of other pathways to compensate for diminished TCA cycle function. Our findings will help guide the rational selection of therapies to overcome drug resistance.Despite the development of metabolism-based therapy for a variety of malignancies, resistance to single-agent treatment is common due to the metabolic plasticity of cancer cells. Improved understanding of how malignant cells rewire metabolic pathways can guide the rational selection of combination therapy to circumvent drug resistance. Here, we show that human T-ALL cells shift their metabolism from oxidative decarboxylation to reductive carboxylation when the TCA cycle is disrupted. The α-ketoglutarate dehydrogenase complex (KGDHC) in the TCA cycle regulates oxidative decarboxylation by converting α-ketoglutarate (α-KG) to succinyl-CoA, while isocitrate dehydrogenase (IDH) 1 and 2 govern reductive carboxylation. Metabolomics flux analysis of T-ALL reveals enhanced reductive carboxylation upon genetic depletion of the E2 subunit of KGDHC, dihydrolipoamide-succinyl transferase (DLST), mimicking pharmacological inhibition of the complex. Mechanistically, KGDHC dysfunction causes increased demethylation of nuclear DNA by α-KG-dependent dioxygenases (e.g., TET demethylases), leading to increased production of both IDH1 and 2. Consequently, dual pharmacologic inhibition of the TCA cycle and TET demethylases demonstrates additive efficacy in reducing the tumor burden in zebrafish xenografts. These findings provide mechanistic insights into how T-ALL develops resistance to drugs targeting the TCA cycle and therapeutic strategies to overcome this resistance.

Abstract 3365: Transcriptomics-based stratification of response to MAPK inhibition in vitro better predicts sensitivity to single agent and combination treatment than MAPK genomic alteration status alone

Abstract Overactivation of mitogen-activated protein kinase (MAPK) signaling is a frequently observed driver of many cancers. Targeted MAPK pathway inhibitors have been approved as therapies in patient populations with genomic alterations to MAPK pathway genes (e.g. dabrafenib in BRAF-mutant melanoma), with multiple other MAPK-targeted therapies in development. We have previously demonstrated that cell lines with MAPK pathway genomic alterations show increased sensitivity to ASTX029, our potent and selective dual-mechanism ERK inhibitor which is currently undergoing clinical development. However, MAPK oncogene status alone is not a universal indicator of sensitivity, nor is it clear that genomic markers of sensitivity to single agent inhibition represent the optimal approach to predicting response to MAPK targeted agents in the combination setting (e.g. sotorasib + trametinib). We therefore analysed MAPK pathway related transcriptional signatures to assess predictability for response to ASTX029 in combination with a SHP2 inhibitor. We previously presented data from large-scale viability screens assessing the response to ASTX029 as a single agent treatment (437 cell lines), as well as in combination with an inhibitor of SHP2 (491 cell lines), an upstream adaptor protein in the MAPK pathway. Using these data, several published transcriptional signatures related to the MAPK pathway were assessed for their ability to stratify sensitive vs resistant cell lines when benchmarked against MAPK oncogene genomic status alone. All assessed transcriptomics-based scores had a significant positive correlation with growth inhibition for ERK inhibitor single agent treatment as well as in combination with a SHP2 inhibitor. Transcriptomics-based scores were shown to be more sensitive than the use of mutant MAPK oncogene status alone based on assessment of binomial regression models. The significant increase in sensitivity enables the identification of additional cell lines responsive to MAPK pathway inhibition. The sensitivity was further increased when considering response to the combination of an ERK and SHP2 inhibitor compared to the ERK inhibitor as a single agent. This analysis demonstrates the potential use of MAPK pathway-related transcriptomics-based scores for the stratification of MAPK pathway inhibitor response. ASTX029 is currently undergoing clinical development in advanced solid tumours (NCT03520075) and transcriptomics-based methods may provide utility in refining patient selection in the clinic for single agent or combination approaches. Citation Format: Jessica Brothwood, Andrea Biondo, Kim-Hien Dao, Keisha Hearn, Chris Hindley, Navrohit Kandola, Harold Keer, John Lyons, Yoko Nakatsuru, Marc O'Reilly, Nicola Wallis, Harpreet Saini. Transcriptomics-based stratification of response to MAPK inhibition in vitro better predicts sensitivity to single agent and combination treatment than MAPK genomic alteration status alone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3365.

Abstract 2010: Transcriptional antagonism by CDK8 inhibition improves therapeutic efficacy of MEK inhibitors

Abstract Mutations in the RAS/MAPK pathway are frequent drivers of oncogenesis. Clinically approved MEK inhibitors are effectively used to target RAS-pathway driven cancers in combination with RAF inhibitors in BRAF mutant cancers such as melanoma and non-small cell lung cancer. However, single agent treatment with MEK inhibitors is not typically sufficient for clinical benefit. Therefore, combination therapy approaches will be required for maximal efficacy in most advanced cancers. Here, we sought to identify MEK inhibitor-based combination therapy approaches in the setting of RAS-mutant neuroblastoma. Neuroblastoma is a cancer that arises in the developing peripheral nervous system. Children with low-risk disease frequently benefit from therapy, but 50-60% of children with high-risk disease will ultimately relapse, and relapsed disease is generally incurable. We performed genome-scale functional genomic drug modifier screens and identified mediator kinase module members CCNC and CDK8 as two genes that when lost sensitize RAS-mutant NBL to MEK inhibition. We used small molecule inhibitors of CDK8 and found that combined inhibition of MEK and CDK8 leads to an improved therapeutic response in vitro and in vivo in a xenograft model of neuroblastoma. Using transcriptional profiling, we established that CDK8 loss and MEK inhibition induce opposite transcriptional signatures. When combined, CDK8 loss prevents the compensatory upregulation of the pro-growth gene expression program induced by MEK inhibition, while the downregulation of MAPK signaling is maintained. Finally, we show that this combination is effective in a subset of other RAS-mutant cancers including pancreatic and lung cancers. Together these data suggest that the mediator kinase module is critical for the adaptive pro-survival transcriptional response induced by MEK inhibition in RAS-mutant neuroblastoma and other RAS-driven malignancies, and that the addition of a CDK8 inhibitor may improve clinical response to MEK inhibitors. Our data supports a model where agents with antagonistic transcriptional programs can have synergistic therapeutic effects when combined, which may be a broadly relevant approach in combination therapies. Citation Format: Clare F. Malone, Minjee Kim, Gabriela Alexe, Alexandra B. Forman, Amanda Robichaud, Amy Saur Conway, Amy Goodale, John M. Hatcher, Nathanael S. Gray, Federica Piccioni, Kimberly Stegmaier. Transcriptional antagonism by CDK8 inhibition improves therapeutic efficacy of MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2010.

Abstract 1664: Model characterization and tumor immune profile assessment for syngeneic RM-1 murine prostate cancer in male C57BL/6 mice

Abstract Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. However, advanced prostate cancer is difficult to treat due to a lack of effective approaches for disrupting immune tolerance. RM-1 is a murine prostate cancer cell line derived from a Ras/Myc-induced prostate cancer. RM-1 is of particular interest because it is an aggressive, nonimmunogenic, potentially metastatic prostate line that is androgen independent. Subcutaneous growth kinetics were evaluated for RM-1 cells implanted at two cell inocula (1.0E+06 and 5.0E+05 cells/implant). RM-1 grew aggressively across both implant conditions, producing 100% take rate, a median tumor volume doubling time of ~2 days, and a median time to euthanasia criteria (2000 mm3) of ~11 days. Bioanalysis of tumor-infiltrating lymphocyte (TIL) composition via flow cytometry confirms that the RM-1 tumor model has characteristics of a nonimmunogenic or a “cool” tumor. CD4+ T cell and CD8+ T cell populations ranged from 0.7 to 1.9% of the CD45+ cells. Comparatively, G-MDSC and M-MDSC populations were much higher at 8.3 and 63.8% of CD45+ cells. This type of TIL profile is ideal for combination therapies, providing a clear opportunity to turn a “cool” tumor “warm” and therefore potentially more responsive to treatment. To further characterize the RM-1 model, we evaluated anti-mPD-1, anti-mPD-L1, and anti-mCTLA-4. As expected, based on the TIL profile, single agent treatment with checkpoint inhibitors produced minimal anticancer activity, with no tumor regressions, and &amp;lt;56% punitive responders. We also evaluated the anticancer activity of focal beam radiation treatment via small animal radiation research platform (SARRP). Single agent radiation treatment was highly effective, with 20, 10 and 5 Gy producing, 100, 78 and 89% incidence of putative responders. Radiation treatment at 5 Gy was selected as an ideal candidate for combination therapy. Combination therapy of radiation treatment at 5 Gy paired with anti-mPD-1, was evaluated to determine if there was an increase in activity with a two-prong approach. Historically, radiation treatment has been used to increase tumor infiltration and reduce the immunosuppressive tumor microenvironment, allowing for increased efficacy. Single agent treatment of anti-mPD-1 or radiation treatment (5 Gy) resulted in 20 and 30% incidence of putative responders. Combination therapy of anti-mPD-1 and radiation (5 Gy) resulted in 100% putative responders. Overall, the RM-1 model has proven to be a reliable and reproducible syngeneic tumor model for prostate cancer. Its presumed immunosuppressive microenvironment is clinically relevant and can provide a direct correlation to patient prostate cancer. Future studies will include orthotopic implant optimization with checkpoint inhibitor efficacy evaluation, including TIL analysis. Citation Format: Justin Snider, Derrik Germain, Patrick Allison, Alden Wong, Sylvie Kossodo. Model characterization and tumor immune profile assessment for syngeneic RM-1 murine prostate cancer in male C57BL/6 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1664.

Abstract 4195: Evaluation of the anti-myeloma effects of high and metronomic selinexor alone and in combination with duvelisib in vitro and in vivo

Abstract Introduction: Selinexor (Karyopharm Therapeutics) is an FDA-approved oral selective inhibitor of nuclear export (SINE) compound. It specifically blocks XPO1 enforcing the nuclear retention of tumor suppressor proteins restoring their anti-cancer functions. It also attenuates the phosphoinositide-3 kinase (PI3K)-AKT pathway by activating PTEN. Although activation of PI3K signaling is often detected in hematological malignancies, there are no data regarding the combination of selinexor with the PI3K inhibitor duvelisib for MM treatment. Thus, we evaluated the anti-myeloma effects of selinexor in combination with duvelisib in vitro and also studied alternative schedules of selinexor with dexamethasone in vivo using our MM xenograft models LAGκ-2. Methods: The human MM cell line U266 was obtained from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow (BM) aspirates. Cell viability was quantified using the MTS cell proliferation assay. For the in vivo studies, the human myeloma xenograft LAGκ-2 was surgically implanted into SCID mice. Selinexor was administered via oral gavage at 2.5 mg/kg 5x per week, 5 mg/kg 4x per week on consecutive days, or 10 mg/kg 2x per week with one day between treatments. Dexamethasone was administered via intraperitoneal injection at 4 mg/kg 2x per week except the vehicle control. Results and Discussion: MTS assays showed selinexor induced concentration dependent inhibition of cell viability/proliferation in the MM cell line MM1s at 0.1, 0.4 and 1.6 μM. Selinexor alone at 0.2 μM reduced cell proliferation of primary MM tumor cells obtained from BM mononuclear cells (MCs) from a patient with progressive disease. We next examined cell viability in the presence of selinexor and duvelisib. MM1s or fresh BM mononuclear cells were incubated with a fixed concentration of selinexor (0.1μM) with increasing concentrations of duvelisib (1.5 μM -25 μM) for 48 h. The results showed that duvelisib enhanced the cytotoxic effects of selinexor in a concentration dependent manner. We also evaluated dosing and scheduling of selinexor and dexamethasone in vivo using SCID mice bearing LAGκ-2 MM xenografts. When selinexor was combined with dexamethasone, lower more frequent dosing showed greater inhibition of tumor growth than mid-range and higher less frequent dosing of this SINE. This study illustrates that the combination of selinexor and duvelisib showed enhanced cytotoxic effects in vitro compared with single agent treatment, and we will further evaluate the anti-MM effects of selinexor in combination with duvelisib in vivo using our human MM xenograft models. These studies suggest that the combination of a SINE and PI3K inhibitor may be effective for MM treatment. Citation Format: Haiming Chen, Stacy Behare, Mingjie Li, Marissa Goldwater, Ava Hekmati, Jasmin Cao, Rocky Levin, Ning Xu, James Berenson. Evaluation of the anti-myeloma effects of high and metronomic selinexor alone and in combination with duvelisib in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4195.