Single-agent Treatment Research Articles

Pediatric Non-Myofibroblastic Primitive Spindle Cell Tumors with ALK Gene Rearrangements and Response to Crizotinib.

We describe two poorly differentiated, non-myofibroblastic (SMA-, S100+, CD34±), spindle cell neoplasms with immunohistochemical positivity for ALK and with ALK gene rearrangements leading to PLEKHH2::ALK and CLTC::ALK fusions, respectively. ALK protein overexpression and/or gene fusions should be evaluated in poorly differentiated spindle cell neoplasms, even when there is an absence of a myofibroblastic phenotype. A positive ALK evaluation has therapeutic implications as both tumors responded to single-agent treatment with the tyrosine kinase inhibitor crizotinib.

Restoring erectile function by combined treatment with JNK inhibitor and HDAC inhibitor in a rat model of cavernous nerve injury.

The main pathophysiologic conditions of erectile dysfunction (ED) after radical prostatectomy are considered to be corporal fibrosis and apoptosis induced by cavernosal nerve (CN) injury. In a rat model of CN crush injury (CNCI), we investigated whether combination treatment with JNK inhibitor (JNKi), SP600125, and HDAC inhibitor (HDACi), suberoylanilide-hydroxamic-7 acid (SAHA), for 2weeks after CNCI would restore erectile function by suppressing fibrosis and apoptosis through normalization of JNK and HDAC pathways. Seventy 12-week-old rats were randomly divided into five groups: Sham surgery, CNCI alone, CNCI treated with daily intraperitoneal injection of 10mg/kg JNKi, CNCI treated with daily oral administration of 25.0mg/kg HDACi, and CNCI daily treated with a combination. Two weeks after CNCI, we investigated the erectile response to electrostimulation and conducted histological staining, caspase-3 activity assay, and western blot analysis. CNCI alone resulted in significantly reduced intracavernosal pressure/mean arterial pressure (MAP) and area under the curve/MAP, decreased smooth muscle (SM)/collagen ratio and SM content, higher caspase-3 activity, and increased protein levels of total HDAC3, transforming growth factor (TGF)-β, fibronectin, and c-Jun phosphorylation, compared with the Sham surgery. The CNCI groups exposed to JNKi, HDACi or both showed improvements in erectile-responses and SM/collagen ratio, compared to the CNCI alone. The combined treatment showed additional improvement in erectile responses at 1.0V stimulation and in SM/collagen ratio compared to the single agent treatment. SM content, caspase-3 activity, and c-Jun phosphorylation improved in the two CNCI groups exposed to JNKi. The two CNCI groups exposed to HDACi showed normalization of protein levels of HDAC3, fibronectin, and TGF-β. The combined administration of JNKi and HDACi during the acute phase after CNCI in rats can preserve ED by suppressing cavernosal fibrosis and apoptosis by normalizing the HDAC/TGF-β and JNK pathways.

Phase I study of hydroxychloroquine plus binimetinib in patients with metastatic pancreatic cancer (the HOPE trial).

TPS634 Background: Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with a dearth of effective therapeutic options. Over 90% of PDAC harbor activating mutations in the KRAS oncoprotein, which in turn leads to activation of downstream effector proteins in the the RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signaling cascade serving to promote tumor cell survival, growth and metastasis. Unfortunately, single agent treatment with MAPK-inhibitors have had limited therapeutic efficacy in patients with PDAC owing to development of various tumor-cell intrinsic resistance mechanisms, including upregulation of autophagy. Hydroxychloroquine is an antimalarial drug that functions to inhibit autophagy by inhibiting acidification of lysosomes. Previously published preclinical data suggest combination therapy with binimetinib, a MEK 1/2 inhibitor, and hydroxychloroquine leads to enhanced killing of PDAC cells in vitro and in vivo. Methods: This is a single arm, single center phase I trial of binimetinib plus hydroxychloroquine in patients with metastatic pancreatic cancer harboring a KRAS mutation. All patients will receive binimetinib at a fixed dose of 45mg PO twice daily (14-day cycles) while hydroxychloroquine will be dosed at 400mg PO twice daily (14-day cycles) and dose escalated using a Bayesian optimal interval design with a target toxicity rate of 0.3. Key eligibility criteria include histologically confirmed metastatic pancreatic adenocarcinoma, prior treatment with at least one line of systemic therapy and a documented KRAS mutation. An estimated 24 patients will be enrolled in the first phase of this study and up to 15 patients in the dose expansion cohort. The primary endpoint of this study is to determine the maximum tolerated dose (MTD) of hydroxychloroquine when combined with a fixed dose of binimetinib. Key secondary endpoints include safety and toxicity profile, response rate, progression free survival (PFS) and overall survival (OS). This study is ongoing and has enrolled 10 patients at the time of submission. Clinical trial information: NCT04132505.

Encorafenib, cetuximab, and cytotoxic chemotherapy combinations in BRAFV600E CRC murine models.

145 Background: Based on promising results from latest trials, a crucial point is to evaluate whether encorafenib (E) plus cetuximab (C), alone or in combination with chemotherapy, can improve clinical outcomes relative to current standard of care in previously untreated BRAFV600E mutant mCRC. Considering the high number of BRAFV600E mutant mCRC patients who will never receive a second-line treatment, the rationale of this strategy is to maximize treatment outcome within the first-line setting. Methods: We performed an in vivo study using human BRAFV600E CRC cell line-derived xenografts in nude mice. We evaluated the efficacy of encorafenib (E) + cetuximab (C), FOLFOX, and FOLFIRI, both as individual regimens and in combinations. Mice were treated for 3 weeks and followed for an additional 8 weeks to evaluate durability of tumor control. Additionally, we validated our findings using 3 BRAFV600E mutated patient derived xenografts. Tumors progressing on single agent and combined treatment were profiled by RNA sequencing, protein extraction for RPPA/Western blot, and establishment of in vitro primary cell cultures for further analyses. Results: Our study showed across all 4 models both FOLFOX and FOLFIRI, each in combination with encorafenib plus cetuximab, having greater efficacy than encorafenib plus cetuximab or either chemotherapy alone. No significant change in toxicity was seen with the addition of chemotherapy. Interestingly, in the one model with long term treatment, FOLFOX + E +/- C performed greatest over the long-term, with significant endpoint separation against all other treatment arms (P < 0.05). Conclusions: Taken together, results from our study suggest that the addition of chemotherapy to BRAF+EGFR targeted therapy can further increase the magnitude of response in BRAFV600E mCRC and is a promising combination now being explored clinically. Additionally, this research will substantially contribute to our understanding of the genetic and molecular bases of resistance to target therapies and chemo-based approach in BRAFV600Econtext.

CPNE1 promotes non-small cell lung cancer progression by interacting with RACK1 via the MET signaling pathway

BackgroundNon-small cell lung cancer (NSCLC) is the most common type of lung cancer and the most lethal tumour worldwide. Copine 1 (CPNE1) was identified as a novel oncogene in NSCLC in our previous study. However, its specific function and relative mechanisms remain poorly understood.MethodsThe biological role of CPNE1 and RACK1 in NSCLC was investigated using gene expression knockdown and overexpression, cell proliferation assays, clonogenic assays, and Transwell assays. The expression levels of CPNE1, RACK1 and other proteins were determined by western blot analysis. The relationship between CPNE1 and RACK1 was predicted and investigated by mass spectrometry analysis, immunofluorescence staining, and coimmunoprecipitation. NSCLC cells were treated with a combination of a MET inhibitor and gefitinib in vitro and in vivo.ResultsWe found that CPNE1 facilitates tumorigenesis in NSCLC by interacting with RACK1, which further induces activation of MET signaling. CPNE1 overexpression promoted cell proliferation, migration, invasion and MET signaling in NSCLC cells, whereas CPNE1 knockdown produced the opposite effects. In addition, the suppression of the enhancing effect of CPNE1 overexpression on tumorigenesis and MET signaling by knockdown of RACK1 was verified. Moreover, compared to single-agent treatment, dual blockade of MET and EGFR resulted in enhanced reductions in the tumour volume and downstream signaling in vivo.ConclusionsOur findings show that CPNE1 promotes tumorigenesis by interacting with RACK1 and activating MET signaling. The combination of a MET inhibitor with an EGFR-TKI attenuated tumour growth more significantly than either single-drug treatment. These findings may provide new insights into the biological function of CPNE1 and the development of novel therapeutic strategies for NSCLC.3n8P5VYzQLauDz5o6kqwUEVideo

Vitamin D3 and zinc synergistically induce regulatory T cells and suppress interferon-γ production in mixed lymphocyte culture

Although vitamin D3 (cholecalciferol) and zinc are known to individually shift the immune response towards tolerance, little is known about the effect of their combined administration. This work contributes to understanding the combined action of zinc and vitamin D3 in different in vitro models for immune reactions. Zinc and vitamin D3 in combination boosted the differentiation into Foxp3+CD4+ T cells (Treg). Vitamin D3 alone reduced the percentage of CD4+T-bet+ T cells (TH1). In mixed lymphocyte culture (MLC), therapeutic concentrations of vitamin D3 and zinc in combination suppressed interferon-γ (IFN-γ) secretion more strongly than the single agent treatment. This effect was also detected for a combination of subtherapeutic concentrations of both vitamin D3 and zinc. Vitamin D3, even at nanomolar concentrations, increased intracellular zinc levels. PBMC (peripheral blood mononuclear cells) of individuals at risk of zinc deficiency responded to vitamin D3 treatment with a higher mRNA expression of Zip13. In PBMC, both agents reduced activation-induced IL-17 secretion. In summary, this study shows, for the first time, a vitamin D3-induced upregulation of CD4+Foxp3+ T cells in MLC. The data propose a model where zinc augments the effect of vitamin D3 in certain therapeutic and subtherapeutic concentrations. Lower concentrations of both vitamin D3 and zinc could be used for effective treatment, thus reducing possible side effects from vitamin D3 and zinc. Vitamin D3 and zinc in combination may be a promising and cheap option to treat dysregulated immune response in various conditions.

Transcriptomics reveals in vivo efficacy of PARP inhibitor combinatorial synergy with platinum-based chemotherapy in human non-small cell lung carcinoma models.

Inhibitors of poly(ADP)-ribose polymerase (PARP) exploit defective DNA repair pathways existing in several forms of cancer, such as those with BRCA mutations, and have proven clinical efficacy as chemosensitizers. However, platinum-based chemopotentiation by PARP inhibitors (PARPi), particularly for non-small cell lung cancer (NSCLC), has only been confirmed in a few preclinical models and the molecular mechanisms that drive PARPi combinatorial synergy with chemotherapeutics remains poorly defined. To better understand these mechanisms, we characterized cisplatin and veliparib efficacy in A549 and Calu6 NSCLC in vivo tumor xenograft models and observed combinatorial synergy in the Calu6 model. Transcriptome-wide analysis of xenografts revealed several differentially expressed genes (DEGs) between untreated and cisplatin + veliparib-treated groups, which were unique from genes identified in either of the single-agent treatment arms. Particularly at 10- and 21-days post-treatment, these DEGs were enriched within pathways involved in DNA damage repair, cell cycle regulation, and senescence. Furthermore, TGF-β- and integrin-related pathways were enriched in the combination treatment arm, while pathways involved in cholesterol metabolism were identified at earlier time points in both the combination and cisplatin-only groups. These data advance the biological underpinnings of PARPi combined with platinum-based chemotherapy and provides additional insight into the diverse sensitivity of NSCLC models.

Onvansertib and paclitaxel combined in platinum-resistant ovarian carcinomas.

Background:Ovarian carcinoma is extremely sensitive to (platinum-based) chemotherapy; however, most patients will relapse with platinum-resistant disease, badly affecting their prognosis. Effective therapies for relapsing resistant tumors are urgently needed.Methods:We used patient-derived xenografts (PDXs) of ovarian carcinoma resistant to cisplatin (DDP) to test in vivo the combination of paclitaxel (15 mg/kg i.v. once a week for 3 weeks) and onvansertib, a plk1 inhibitor, (50 mg/kg orally 4 days a week for 3 weeks). The PDX models were subcutaneously (s.c.) or orthotopically transplanted in nude mice and antitumor efficacy was evaluated as tumor growth inhibition and survival advantages of the combination over untreated and single agent treatment.Results:The combination of onvansertib and paclitaxel was very well tolerated with weight loss no greater than 15% in the combination group compared with the control group. In the orthotopically transplanted PDXs, single onvansertib and paclitaxel treatments prolonged survival; however, the combined treatment was much more active, with median survival from three- to six-fold times that of untreated mice. Findings were similar with the s.c. transplanted PDX, though there was greater heterogeneity in tumor response. Ex vivo tumors treated with the combination showed greater induction of γH2AX, marker of apoptosis and DNA damage, and pSer10H3, a marker of mitotic block.Conclusion:The efficacy of onvansertib and paclitaxel combination in these preclinical ovarian cancer models supports the clinical translatability of this combination as an effective therapeutic approach for platinum-resistant high-grade ovarian carcinoma.

Epigenetic therapy combination of UNC0638 and CI-994 suppresses breast cancer via epigenetic remodeling of BIRC5 and GADD45A.

There is currently a growing interest in the roles of epigenetic mechanisms in the diagnosis, prognosis, and therapies associated with precision oncology for breast cancer (BC). This study aimed to demonstrate the clinical significance of euchromatic histone lysine methyltransferase 2 (EHMT2), histone deacetylase 1 (HDAC1) and HDAC2 in BC, to evaluate the antitumor effectiveness of a combination of the selective inhibitors UNC0638 and CI-994 (U+C), and to clarify the underlying mechanisms. Multi-omic analysis was used to study the clinical significance of the biomarkers of interest. The effects of U+C treatment were evaluated by detecting cell viability, cell cycle, apoptosis, and representative gene expressions. RNA-Seq and Gene Set Enrichment Analysis (GSEA) were employed to identify over-represented genes associated with the treatment. Chromatin immunoprecipitation and qPCR (ChIP-qPCR) assay were applied to verify epigenetic profiling on the identified promoters. The significance of elevated expressions of EHMT2, HDAC1, and HDAC2 in tumor tissue and BC basal-like subtype in predicting a poor prognosis was noted. The U+C combined treatment showed an enhanced suppressive effect as compared to single agent treatment, perturbed the cell cycle, induced apoptosis, reduced expressions of the genes representing anti-apoptosis, stemness, drug resistance and basal-like state, while increasing luminal-like state genes. In addition, the combined U+C treatment suppressed xenograft tumor growth. The epigenetic reprogramming of histones was identified in the down-regulated BIRC5 and upregulated GADD45A. These findings demonstrate that selectively targeting EHMT2, HDAC1, and HDAC2 by concurrent U+C treatment suppresses BC tumor progression via epigenetic remodeling of BIRC5 and GADD45A.

Morphological Characterization of Antibiotic Combinations.

Combination therapies are common in many therapeutic contexts, including infectious diseases and cancer. A common approach for evaluating combinations in vitro is to assess effects on cell growth as synergistic, antagonistic, or neutral using "checkerboard" experiments to systematically sample combinations of agents in multiple doses. To further understand the effects of antibiotic combinations, we employed high-content imaging to study the morphological changes caused by combination treatments in checkerboard experiments. Using an automated, unsupervised image analysis approach to group morphologies, and an "expert-in-the-loop" to annotate them, we attributed the heterogeneous morphological changes ofEscherichia coli cells to varying doses of both single-agent and combination treatments. We identified patterns of morphological change, including morphological potentiation, competition, and the emergence of unexpected morphologies. We found these frequently did not correlate with synergistic or antagonistic effects on viability, suggesting morphological approaches may provide a distinctive signature of the biological interaction between compounds over a range of conditions. Among the unexpected morphologies we observed, there were transitional changes associated with intermediate doses of compounds and uncharacterized phenotypes associated with well-studied antibiotics. Our approach exemplifies how unsupervised image analysis and expert knowledge can be combined to reckon with complex phenotypic changes arising from combination screening, dose titrations, or polypharmacology. In this way, quantification of morphological diversity across treatment conditions could aid in analysis and prioritization of complementary pairings of antibiotic agents by more closely capturing the true signature of the integrated cellular response.

Preclinical evidence for pioglitazone and bexarotene combination in oral cancer chemoprevention.

Head and neck squamous cell carcinoma (HNSCC) requires new treatments and targeted approaches to improve survival. The peroxisome proliferator-activated receptor γ (PPARγ) and retinoic X receptor alpha (RXRα) nuclear receptor pathways may be targetable with repurposed Food and Drug Administration (FDA)-approved agents for prevention and treatment. Oral cancer and leukoplakia cell lines were treated with the PPARγ agonist (pioglitazone) and RXRα activator (bexarotene). PPARγ activation, cellular proliferation, apoptosis activity and phenotype, including the pharmacodynamic marker, involucrin (IVL), were subsequently analyzed using a reporter gene assay, genomic data, MTT assay and western blot. Microarray analysis of HNSCC tumor versus normal tissue shows IVL expression is significantly increased in normal tissue compared to HNSCC tumors (p < 0.0001). In MSK Leuk1 and CA 9-22 cell lines, pioglitazone increases PPARγ DNA binding activity and IVL promoter activity in a dose dependent manner (p < 0.01 and p < 0.0001). Combination treatment with pioglitazone and bexarotene increases PPARγ DNA binding activity and IVL promoter activity (p < 0.01 and p < 0.0001). MTT analysis shows decreases in cell proliferation when cells are treated with pioglitazone and bexarotene. Decreases in cell proliferation are significant to at least p < 0.05 for all combination versus single agent treatments. Western blot on whole-cell lysate from cells treated with pioglitazone and bexarotene alone or in combination for IVL showed increased protein levels with combination treatment. Targeting the PPARγ/RXRα heterodimer with pioglitazone and bexarotene was effective in this preclinical project. This was functional in both preneoplastic and oral cancer cell lines. A better understanding of the molecular mechanism on downstream effects on cellular proliferation could potentially have implications clinically, both in oral preneoplasia and possibly head and neck cancer; however, more research needs to be done to explore the potential these medications have in chemoprevention.

Efficacy Evaluation of Plant Products in the Treatment of Erectile Dysfunction Related to Diabetes.

Erectile dysfunction affects more than 50% of diabetic male patients, with a higher prevalence compared with the general population. Age, clinical factors, and lifestyle habits have been suggested to contribute to the pathophysiology and worsening of erectile dysfunction in diabetic patients. First- and second-line standard treatments are represented by phosphodiesterase type 5 (PDE5) inhibitors and alprostadil, respectively. However, natural compounds have been suggested to ameliorate this clinical condition. This study aims to preclinically characterize the potential synergism among plant-derived products for the improvement of erectile dysfunction in the diabetic condition. The effects of a nutritional supplement composed of Panax ginseng, Moringa oleifera and rutin, as single agents or as a mixture, were evaluated in a streptozotocin (STZ)-induced diabetic rat model with erectile dysfunction. The treatment efficacy was evaluated by measuring sexual-related parameters (i.e., mount and intromission latencies, the mount and intromission frequencies and the ejaculation latency). Results showed that only the mixture was able to significantly reduce the diabetes-related delay in mount latency (p < 0.01). Substantial similar effects were observed by measuring the intromission latency and the mean number of mounts was very similar between rats treated with the mixture and controls. Single agent treatments showed very low effects in terms of intromission frequency, whereas the mixture was able to increase this parameter. Additionally, a statistically significant reduced ejaculation latency was observed in rats treated with the mixture compared with the STZ control. These results are in agreement with the available literature and suggest that the study mixture may ameliorate sexual behavior compared with the administration of the study natural compounds as single agents in diabetic rats. Further preclinical and clinical studies are needed to perform a more comprehensive evaluation of the efficacy and safety of the study mixture.

Enhancement of Radiosensitivity by DNA Hypomethylating Drugs through Apoptosis and Autophagy in Human Sarcoma Cells

The targeting of DNA methylation in cancer using DNA hypomethylating drugs has been well known to sensitize cancer cells to chemotherapy and immunotherapy by affecting multiple pathways. Herein, we investigated the combinational effects of DNA hypomethylating drugs and ionizing radiation (IR) in human sarcoma cell lines both in vitro and in vivo. Clonogenic assays were performed to determine the radiosensitizing properties of two DNA hypomethylating drugs on sarcoma cell lines we tested in this study with multiple doses of IR. We analyzed the effects of 5-aza-dC or SGI-110, as DNA hypomethylating drugs, in combination with IR in vitro on the proliferation, apoptosis, caspase-3/7 activity, migration/invasion, and Western blotting using apoptosis- or autophagy-related factors. To confirm the combined effect of DNA hypomethylating drugs and IR in our in vitro experiment, we generated the sarcoma cells in nude mouse xenograft models. Here, we found that the combination of DNA hypomethylating drugs and IR improved anticancer effects by inhibiting cell proliferation and by promoting synergistic cell death that is associated with both apoptosis and autophagy in vitro and in vivo. Our data demonstrated that the combination effects of DNA hypomethylating drugs with radiation exhibited greater cellular effects than the use of a single agent treatment, thus suggesting that the combination of DNA hypomethylating drugs and radiation may become a new radiotherapy to improve therapeutic efficacy for cancer treatment.

Resolution of systemic complications in Schistosoma mansoni-infected mice by concomitant treatment with praziquantel and Schisandrin B

Schistosomiasis is a tropical parasitic disease, in which the major clinical manifestation includes hepatosplenomegaly, portal hypertension, and organs fibrosis. Clinically, treatment of schistosomiasis involves the use of praziquantel (PZQ) and supportive care, which does not improve the patient’s outcome as liver injuries persist. Here we show the beneficial effects of using PZQ in combination with Schisandrin B (Sch B). Concomitant treatment with PZQ and Sch B resulted in a significant improvement of hepatosplenomegaly and fibrosis, compared with single-agent treatment. We also demonstrated that PZQ-Sch B treatment ameliorates injuries in the lungs and intestine better than the sole use of PZQ or Sch B. In addition, PZQ-Sch B treatment improves the survival of S. mansoni-infected mice, and the treatment combination yields better therapeutic outcomes, as indicated by a partial improvement in neurological function. These results were accompanied by a reduction in neurological injuries. Collectively, we suggest that PZQ-Sch B concomitant therapy may be useful to alleviate schistosomiasis-associated liver injuries and prevent systemic complications.

Abstract P084: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with EGFR inhibitors osimertinib and afatinib in NSCLC preclinical models

Abstract Background: Activating mutations in EGFR have been reported in ~30% of patients with non-small cell lung cancer (NSCLC). Three generations of small molecule EGFR kinase inhibitors have been approved by the FDA to treat these patients, however, multiple mechanisms of resistance cause cancer progression. In addition to drug-resistant mutations that arise and re-activate EGFR, other signaling pathways can be activated to cause resistance. Although EGFR inhibitors such as osimertinib and afatinib (also a pan-ErbB inhibitor) have demonstrated clear clinical benefits, patients inevitably develop resistance. Herein we demonstrate mutant EGFR NSCLCs activate autophagy upon treatment with EGFR inhibitors as a drug resistance mechanism. Therefore, a combination of an EGFR inhibitor with an autophagy inhibitor has the potential to deepen and prolong responses and improve patient outcomes. Materials and Methods: Human cancer cells with EGFR mutations were cultured using recommended complete medium. Inhibition of ULK1/2 was measured through standard biochemical assays and cellular readouts including NanoBRET and ELISA-based ATG13 phosphorylation assays. Autophagosome formation was measured using the CytoID assay (Enzo Life Sciences). For in vivo studies, NCI-H1975 cells that harbor an EGFR T790M resistance mutation were inoculated into BALB/c nude mice. Statistical analyses for the in vivo studies were performed using the Student’s t-test. Results: Erlotinib, gefitinib, osimertinib, and afatinib activated autophagy 3–4-fold over basal levels in the HCC827 cell line (EGFR exon 19 deletion) as measured by increases in phosphorylated ATG13, a cellular substrate of the autophagy-initiating kinases ULK1/2. DCC-3116, an investigational potent and selective dual inhibitor of ULK1 (IC50 6 nM) and ULK2 (9 nM) in cellular assays, inhibited both EGFR-induced and basal phosphorylation of ATG13 with IC50 values of 61–66 nM. Treatment of the NCI-H1975 EGFR mutated (L858R/T790M) NSCLC cell line with osimertinib or afatinib induced autophagy 3-fold over basal levels. DCC-3116 potently inhibited osimertinib and afatinib induced phosphorylation of ATG13 with IC50 values of 91 nM and 71 nM, respectively, and inhibited the increase in autophagosomes induced by these agents. Importantly, these in vitro effects translated to in vivo efficacy. The combination of DCC-3116 with osimertinib or afatinib resulted in significantly greater tumor responses than single agent treatments in the NCI-H1975 NSCLC xenograft model (combination vs. osimertinib p = 0.0005; combination vs. afatinib p = 0.0001; osimertinib combination vs vehicle p &amp;lt; 0.0001; afatinib combination vs. vehicle p &amp;lt; 0.0001). These data provide a strong rationale to study the combination of the ULK inhibitor DCC-3116 with EGFR inhibitors such as osimertinib and afatinib in cancer patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors with a documented RAS or RAF mutation (NCT04892017). Citation Format: Madhumita Bogdan, Mary J. Timson, Hikmat Al-Hashimi, Yu Zhan, Bryan D. Smith, Daniel L. Flynn. DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with EGFR inhibitors osimertinib and afatinib in NSCLC preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P084.

Abstract P029: CDC7 inhibitor-induced replication stress generates inflamed aneuploid cells to sensitize immune checkpoint inhibitors

Abstract Background: Cell division cycle 7 (CDC7), a serine/threonine kinase, plays important roles in the initiation of DNA replication. We developed a highly specific CDC7 inhibitor, TAK-931, as a next-generation of replication stress (RS) inducer. In this study, we preclinically investigated novel aspects of TAK-931 on antitumor efficacy and immunity to evaluate the therapeutic potential of TAK-931 with immune checkpoint inhibitors (ICIs). Methods: TAK-931-treated HeLa cells were subjected to cell growth assay, senescence-associated galactosidase (SA-b-gal) activity assay, transcriptome analysis (RNA-seq), and single-cell RNA-seq (scRNA-seq). The flowcytometry (FCM)-based immune profiling panel studies in J558 allograft syngeneic mouse models were performed at Shanghai Medicilon Inc. In vivo efficacy studies in J558 allograft models in combination with anti-mPD-1, anti-mPD-L1, and anti-mCTLA-4 antibodies were performed at Shanghai Medicilon Inc. Results: TAK-931 intensively induced RS to consequently generates senescence-associated secretory phenotype (SASP) aneuploid cells, which highly expressed inflammatory cytokines and chemokines. In transcriptome analyses, inflammatory cytokine and chemokine hallmarks were also significantly and intensively enriched in the TAK-931-induced aneuploid cells; 5 out of top-6 enriched hallmarks were inflammatory related. The scRNA-seq analyses revealed that advanced aneuploidy was closely associated with activations in the inflammatory-related and SASP-associated pathways. The FCM-based immune profiling panel studies demonstrated that the tumor infiltrating immune cells (TIICs), such as CD8+ T cells, CD4+ T cells, PD-1+CD8+ T cells, and PD-1+CD4+ T cells, were significantly accumulated in TAK-931-treated J558 mouse allografts, while immune suppressive CD45+ myeloid derived suppressor cells (MDSCs) were significantly decreased. Single-agent treatment with TAK-931 exhibited significant antitumor efficacy and immunity in J558 syngeneic allografts, which confirmed by tumor re-challenging studies. We finally demonstrated that combination treatment with TAK-931 and ICIs (anti-mPD-1, anti-mPD-L1, and andti-mCTLA-4 antibodies) enhances antiproliferative activities in preclinical syngeneic mouse models. Conclusions: These preclinical findings suggest the therapeutic potential of TAK-931 in antitumor efficacy and immunity, which may improve clinical benefit of the currently-used immunotherapy by combination treatment. Citation Format: Tomoko Y. Morita, Jie Yu, Yukie Kashima, Kosuke Tanaka, Yumi Hakozaki, Shun-ichiro Kageyama, Akito Nakamura, Eric Lightcap, Huifeng Niu, Karuppiah Kannan, Akihiro Ohashi. CDC7 inhibitor-induced replication stress generates inflamed aneuploid cells to sensitize immune checkpoint inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P029.

Abstract IAP0301: Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities

Abstract T cell-engaging bispecific antibody constructs (TCEs) have the potential to engage all cytotoxic T cells for redirected cancer cell lysis, including CD8, CD4, gamma/delta, NKT and regulatory T cells. TCEs transiently connect T cells with target cells leading to formation of a cytolytic synapse. Once activated by TCEs, T cells can adopt a serial lysis mode and increase troop size by their local proliferation. In numerous animal models, TCEs can eradicate large tumors as single-agent therapy. TCEs show a remarkable single-agent activity in hematological diseases, including ALL, NHL (DLBCL and FL) and AML. A CD19/CD3-bispecific TCE called blinatumomab (Blincyto) has been approved in 2014, and six other TCEs targeting CD20, BCMA and CD123 are in late-stage development. With complete response rates &amp;gt;50% and durability of responses, TCEs are in par with autologous CD19 CAR-T cells in NHL but are easier to manufacture and allow for a faster treatment start. Treatment of solid tumor indications with TCEs is not as successful as treatment of blood-borne cancers. This may be due to issues with tumor penetration, the hostile tumor microenvironment and the scarcity of T cells in cold tumors. A variety of TCEs explore their single-agent activity against solid tumors by targeting surface antigen like PSMA, GPC3, GD2, or HER-2. Enhancements in the form of combination therapy with checkpoint inhibitors, or 4-1BB and CD28 agonists are being explored as ways of augmenting solid tumor activity of TCEs. The first TCE showing robust anti-tumor activity in a solid tumor indication is targeting a gp100 peptide/MHC (developed by Immunocore). It is made from a soluble TCR fragment of very high affinity for the gp100 peptide HLA*0201 complex fused to an anti-CD3 scFv fragment. Although only few partial responses were observed in uveal melanoma patients, the single-agent treatment showed a very robust improvement in overall survival with a hazard ratio of 0.51 compared to investigators’ choice. This data will inspire the development of more TCEs that are specific for peptide/MHC complexes. This class of targets can also be addresses by TCR-mimetic antibodies. TCEs using such antibodies showed high anti-tumor activity in mouse models against MAGE-A4, p53 mutant or tyrosinase peptide/MHC complexes, all targets that are otherwise not amenable to classical TCEs. Another emerging opportunity for treating cancers are conditional TCEs. These are infused as inactive precursors. Inside tumors, the locally produced proteases convert the precursors into highly active TCEs. One example is the so called ProTriTACs, where a half-life extending albumin-binding domain masks the CD3-binding domain. The mask confers a 500-fold reduction in the cytolytic activity of the TCE. Inside tumors, the mask is cleaved off and a TCE is activated that has a very short serum half-life once it is released from the tumor. Conditional TCEs promise not only a wider therapeutic index but can also leverage as targets tumor-associated antigens that are widely expressed on tumors but also on healthy normal tissues, such as EGFR or EpCAM/TROP1. On-target toxicity of TCEs relates to the quality of target antigens. Finding more tumor-specific surface antigens, appropriate pMHC targets (e.g., mutant oncogene epitopes) and conditional T cell engagers are ways to address this challenge. Off-target toxicity of TCEs relates to T cell activation because of redirected target cell lysis. In the worst case, a cytokine release syndrome is induced. In the best case, a low-level cytokine release is contributing to the efficacy of TCEs. CRS can be managed by steroids, step dosing, anti-IL6 antibody and/or dasatinib. Another promising means is subcutaneous administration of TCEs. A dozen TCE programs currently explore this route, which allows reduction of Cmax and of the initial cytokine release. TCEs will soon become a major weapon in the arsenal of cancer therapy. They will not only work in blood-borne cancers but will soon conquer select solid tumor indications. Peptide/MHC-targeting and conditional TCEs are very promising approaches to achieve solid tumor activity of TCEs. Citation Format: Patrick A. Baeuerle. Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr IAP0301.

Systematic review and meta-analysis of the predictive power of MTHFR polymorphisms for pemetrexed drug efficacy and toxicity in non-small cell lung cancer patients

We conducted a meta-analysis to determine if MTHFR polymorphisms are effective biomarkers for non-small cell lung cancer (NSCLC) patient survival and pemetrexed (PEM) treatment toxicity. Because of data heterogeneity, fixed or random effects models were chosen, and pooled HRs and 95% confidence intervals (CIs) were calculated. No correlation between MTHFR 677 C > T polymorphism and progression-free survival (PFS) or overall survival (OS) was detected in NSCLC patients; however, patients with the T allele benefited more than those with the wild-type allele. Two papers reported hematologic toxicity of single-agent PEM treatment in patients with the MTHFR 677 C > T polymorphism. However, data on MTHFR polymorphisms and toxicity could not be combined, even though publication bias and sensitivity analysis results were stable and reliable. We conclude that the MTHFR 677 C > T polymorphism could not predict PEM efficacy in NSCLC patients; however, the T allele may increase the risk of haematological toxicity. A large-scale clinical trial is recommended.

Inhibitors of Bcl-2 and Bruton\u2019s tyrosine kinase synergize to abrogate diffuse large B-cell lymphoma growth in vitro and in orthotopic xenotransplantation models

Numerous targeted therapies have been developed for diffuse large B-cell lymphoma, but the results of late-stage clinical trials were mostly disappointing and have led to very few new regulatory approvals. Here, we use single and combinatorial drug response profiling to show that the combined inhibition of the anti-apoptotic protein Bcl-2 and of the tyrosine kinase BTK with the small molecules venetoclax and ibrutinib efficiently kills DLBCL cells in vitro. High Bcl-2 expression due to either BCL2 amplifications or translocations, in conjunction with chronic active BCR signaling accurately predict responses to dual Bcl-2/BTK inhibition. Orthotopic xenotransplantation and patient-derived xenograft models confirm that the combinatorial is superior to single-agent treatment in reducing the lymphoma burden. Combinatorial treatment further efficiently overcomes both primary and acquired resistance to venetoclax, which we could link to reduced expression of the Bcl-2 family members Bcl-XL and Bcl-2A1 under ibrutinib. We found in a Swiss DLBCL cohort that ~15% of patients are projected to respond to the venetoclax/ibrutinib combination based on their high Bcl-2 expression and nuclear NF-κB localization. Our data show that drug sensitivities exposed by drug response profiling can be attributed to specific mutational signatures and immunohistochemical biomarkers, and point to combined Bcl-2/BTK inhibition as a promising therapeutic strategy in DLBCL.

IMMU-13. COMBINED MODULATION OF THE TGF-Β PATHWAY AND GITR IMMUNE CHECKPOINT SIGNALING PROMOTES ANTI-TUMOR IMMUNITY IN SYNGENEIC GLIOMA MODELS

Abstract The profound local immunosuppressive microenvironment is one hallmark of glioblastoma, which results in resistance to most immunotherapeutic strategies that have been explored so far. Reverting this condition in order to reinvigorate anti-glioma immunity might be a promising therapeutic approach. Transforming growth factor (TGF)-β signaling is deregulated in different cancer types and contributes to the malignant phenotype of glioma cells. Glioma-derived TGF-β is also a major immunosuppressive factor in the tumor microenvironment. Furthermore, intratumoral regulatory T (Treg) cells and activated T effector cells express high levels of the co-stimulatory immune checkpoint glucocorticoid-induced tumor necrosis factor receptor (GITR). Agonistic anti-GITR antibodies have been explored in preclinical tumor models and are under investigation in clinical trials for the treatment of solid tumors. We evaluated the effect of TGF-β and GITR targeting on anti-tumor immune responses in syngeneic mouse glioma models. In co-culture settings, GITR modulation with a GITR ligand (GITRL)-Fc fusion protein, given alone or in combination with a pharmacological TGF-β receptor inhibitor, led to increased T cell activation. Furthermore, the combined targeting of the two pathways resulted in significantly higher immune cell-mediated tumor cell killing than either treatment alone. In vivo, TGF-β inhibition and GITR signaling modulation resulted in a higher fraction of long-term surviving glioma-bearing mice than single-agent treatment. Surviving mice were resistant to tumor re-challenge, suggesting adaptive immunity as an underlying mechanism. These data support the assumption that combined immunotherapeutic strategies may represent a promising approach for the treatment of glioma.