bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1033: The anticancer compound avicin D targets lysosomal functions
Authors: Braig Friederike , Gutterman Jordan U. , Acosta-Alvear Diego
Published: July 2021

This study investigates the mechanism by which avicins—plant-derived triterpenes with potent anticancer activity—alter lysosomal functions. The anticancer properties of avicins have been known for over two decades, yet the precise mechanism of action of avicins remains unknown. To elucidate their mechanism of action, we conducted a pooled CRISPR interference (CRISPRi) genetic screen in Jurkat T-cell leukemia cells treated with avicin D. Our screen revealed that the depletion of genes encoding lysosomal components protected cells from the drug's cytotoxic effects. Consistent with this finding, blocking lysosomal functions with chloroquine protected cells from avicins. Moreover, blocking the lysosomal cholesterol transporter NPC1 abolished avicin cytotoxicity, hinting that avicins—which resemble steroidogenic molecules—use the same cellular factors required for cholesterol transport. Together, these results suggest a causal relationship between a requirement for lysosomal functions and avicin cytotoxicity. The top hit whose depletion sensitized cells to avicin D was the endoplasmic reticulum (ER) stress sensor ATF6, which is a membrane-tethered transcription factor capable of detecting sphingolipid lipotoxicity in the ER membrane. Consistent with this result, recent observations indicate that avicins alter the cell's sphingolipid and cholesterol balance (1), suggesting that avicins induce ER stress by disrupting ER membrane homeostasis. Together, our results support a potential mechanism of action wherein avicins are pro-drugs activated in lysosomes and induce organelle stress by altering the cell's lipid balance. We are currently working on elucidating the molecular mechanisms by which avicins and their putative metabolic products disrupt organelle homeostasis. We anticipate that our study will reveal the molecular circuitry that controls the cell's response to avicins and may open new avenues for therapeutic intervention in cancer.

1. Garrido CM, Henkels KM, Rehl KM, Liang H, Zhou Y, Gutterman JU, Cho K-J. Avicin G is a potent sphingomyelinase inhibitor and blocks oncogenic K- and H-Ras signaling. PMC7272413

Citation Format: Friederike Braig, Jordan U. Gutterman, Diego Acosta-Alvear. The anticancer compound avicin D targets lysosomal functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1033.
Abstract 1041: Targeted inhibition of c-MET, Axl and DDR signaling pathways by merestinib enhances gemcitabine/nab-paclitaxel standard chemotherapy activity in pancreatic cancer models
Authors: Shi Eda , Schwarz Margaret A. , Hassan Md Sazzad , von Holzen Urs , Schwarz Roderich E. , Awasthi Niranjan
Published: July 2021

Background: Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies in Western countries. Gemcitabine (Gem) plus nab-paclitaxel (NPT) is the standard chemotherapy treatment for PDAC leading to an average survival of 8.5 months. Activated signaling of c-MET, Axl and DDR1/2 pathways have been implicated in tumorigenic growth in many cancers including PDAC. Merestinib (Mer) is a potent, multikinase inhibitor that blocks c-MET, Axl and DDR1/2 pathways with IC50 values in the low nanomolar range. In this study, we investigated the antitumor potency of merestinib to enhance standard chemotherapy response in preclinical PDAC models.

Methods: In vitro cell proliferation assays of PDAC-associated cells were evaluated by colorimetric WST-1 reagent. The mechanism of action was determined by Immunoblot analysis. Tumor growth studies were conducted in 6-week-old female NOD/SCID mice in subcutaneous xenografts.

Results: In human PDAC AsPC-1 cell-derived subcutaneous xenografts, gemcitabine plus nab-paclitaxel and merestinib inhibited tumor growth while their combination had an additive effect. The net increase in tumor size was 357 mm3 in controls, 102 mm3 after Gem+NPT, 177 mm3 after Mer and 66 mm3 after Gem+NPT+Mer. Gemcitabine plus nab-paclitaxel and merestinib suppressed in vitro cell proliferation of PDAC epithelial cells and stromal cells. Importantly, the combination treatment demonstrated additive inhibitory effects. Inhibition in cell proliferation by Gem+NPT, Mer and Gem+NPT+Mer treatments was 54%, 14% and 82% (AsPC-1 cells, at medium dose levels), 54%, 4% and 73% (PANC-1 cells, at the highest dose level), 55%, 58% and 92% (PDAC-associated fibroblasts, at the medium dose level). Immunoblot analysis revealed that merestinib caused a decrease in the expression of phospho-c-MET, phospho-Axl, phospho-IGF-1R, phospho-AKT and an increase in apoptosis-related protein cleaved caspase-3 in PDAC cells either alone or in combination with gemcitabine plus nab-paclitaxel.

Conclusion: Simultaneous inhibition of c-MET, Axl and DDR1/2 pathways by merestinib has antitumor efficacy in PDAC and it can significantly improve the standard chemotherapy response. This therapeutic approach might lead to a clinically relevant combination to increase PDAC patients' survival.

Citation Format: Eda Shi, Margaret A. Schwarz, Md Sazzad Hassan, Urs von Holzen, Roderich E. Schwarz, Niranjan Awasthi. Targeted inhibition of c-MET, Axl and DDR signaling pathways by merestinib enhances gemcitabine/nab-paclitaxel standard chemotherapy activity in pancreatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1041.
Abstract 1046: Synergistic cytotoxic effect of sulfated glycolipid PG545 in combination with ATR inhibition in ovarian cancer cells
Authors: Xiao Yinan , Deng Chaolin , Guan Ye , Jin Ling , Ray Upasana , Thirusangu Prabhu , Staub Julie , Xu Haotian , Shridhar Viji
Published: July 2021

Despite the advances in the molecular understanding of ovarian cancer (OC), improvements in therapy are still needed. PG545 is a highly sulfated small molecule and is currently in Phase I clinical investigation, which has shown promising antitumor activity in multiple tumor models, including ovarian, endometrial and pancreatic cancers. Ataxia telangiectasia and Rad3-related protein (ATR), plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. Several ATR inhibitors have been developed, but resistance is likely to occur. This study aimed to investigate the potential synergistic effect of combining PG545 with ATR inhibition in OC. Proliferation and clonogenic assay showed that combination of PG545 with ATR shRNA or ATR inhibitor (ATRi; VE-822) synergistically decreased cell survival compared with single-agent treatments in two OC cell lines, OVCAR5 and OVCAR8 cells. PG545 monotherapy induced DNA damage, activated the ATR signaling pathways as well as the S and G2/M cell cycle checkpoints. The combination of PG545 and ATRi inhibited the phosphorylation of ATR and Chk1, induced γH2AX and lead to a marked increase in pan-nuclear γH2AX-positive cells. Moreover, ATRi diminished the cell cycle effects of PG545 and markedly enhanced PG545-induced apoptosis in OC cell lines. Taken together, our results indicate that the combination of PG545 with ATR inhibition may be promising options to be considered in future clinical trials of OC.

Citation Format: Yinan Xiao, Chaolin Deng, Ye Guan, Ling Jin, Upasana Ray, Prabhu Thirusangu, Julie Staub, Haotian Xu, Viji Shridhar. Synergistic cytotoxic effect of sulfated glycolipid PG545 in combination with ATR inhibition in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1046.
Abstract 1050: Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia
Authors: Chen Xiaoyue , Caenepeel Sean , Belmontes Brian , McElroy Patricia L. , Rex Karen , Osgood Tao , Hughes Paul
Published: July 2021

Acute Myeloid Leukemia (AML) is the most common and aggressive acute leukemia in adults, with a 25% 5-year survival rate. FLT3 is the most frequently mutated gene in AML. About 25% of AML patients harbor FLT3 internal tandem duplication (ITD) mutations, and about 8 % of patients harbor FLT3 tyrosine kinase domain (TKD) mutations. Both FLT3-ITD and FLT3-TKD mutations constitutively activate the protein, which causes poor survival. Gilteritinib is a highly potent and selective oral FLT3 inhibitor recently approved by the FDA. Although gilteritinib showed strong single agent activity in AML patients with FLT3 mutations, the development of gilteritinib resistance limits the long-term efficacy of this treatment, indicating that combination therapy may be advantageous for AML patients with FLT3 mutations. FLT3 mutations are known to be anti-apoptotic. Myeloid cell leukemia-1 (MCL-1), an anti-apoptotic protein, expressed in a large percentage of the AML patient population, plays a critical role in AML cell survival and drug resistance. AMG 176 is a potent, selective and orally bioavailable MCL-1 inhibitor, which induces rapid commitment to apoptosis in AML. Here we demonstrated that AMG 176 and gilteritinib combination treatment synergistically targeted FLT3-ITD mutated AML. A strong synergistic effect (Combination index <0.2) was observed in two FLT3-ITD mutated AML cell lines treated with the AMG 176 plus gilteritinib combination. Increased Bcl-2-like protein 11 (BIM) expression and decreased BIM/MCL-1 interaction was observed under the combination treatment, coupled with a significant increase in apoptosis. In the MV4-11 AML xenograft model, mice treated with two cycles of AMG 176 (2 days on 5 days off) at 30 mg/kg or with 3 mg/kg gilteritinib daily showed 76% or 74% tumor growth inhibition respectively. A statistically significant increase in efficacy was observed in the combination treatment group, with 92.8% tumor regression, including 50% of mice being tumor free. No significant difference in body weight was found between groups. Our results show that AMG 176 plus gilteritinib treatment was effective and well tolerated in mouse models of AML, which supports further evaluation in a clinical setting.

Citation Format: Xiaoyue Chen, Sean Caenepeel, Brian Belmontes, Patricia L. McElroy, Karen Rex, Tao Osgood, Paul Hughes. Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1050.
Abstract 1054: Patient derived organoids demonstrate synergistic antitumor effect of senogenic and senolytic drug combination of Aurora kinase inhibitor and BCL2/xL inhibitor
Authors: Bharti Vijaya , Weiss Vivian , Shen Chengli , Chen Sheau Chiann , Blevins Ashlyn , Shattuck-Brandt Rebecca L. , Richmond Ann , Vilgelm Anna E.
Published: July 2021

The management of metastatic melanoma continues to be challenging, despite of evolving treatments such as immune checkpoint blockade and inhibitors of mutated BRAF. New therapies that work via immune and BRAF-independent mechanisms are urgently required for tumors that are unresponsive to currently available treatments. Here we hypothesized that combination of Aurora Kinase inhibitor (AURKAi) alisertib (senescence inducer) with a drug that selectively kills senescent cells, such as BCL2/xL inhibitor (BCLi) navitoclax (senolytic), will induce apoptosis in melanoma cells. Patient derived organoids (PDOs) were used to test anti- tumor effect of AURKAi and BCLi combination treatment. H&E staining of PDOs and their matched original tumors revealed similar histology. Fourteen out of ninety PDOs were highly sensitive to AURKAi and BCLi combination treatment exhibiting robust induction of cell death. NextGen DNA sequencing indicated that all sensitive PDOs retained wild type TP53, while resistant tumors had mutations in this gene. Knockout or knockdown of TP53 abrogated AURKAi and BCLi-induced cell death in melanoma cells, suggesting that transcription factor p53 encoded by TP53 is a key mediator of drug response. Addition of BCLi to AURKAi activated proapoptotic p53 targets BAX, NOXA and induced cleavage of PARP and Caspases 3, 7, and 8. Electron microscopy revealed morphological signs of apoptosis in combination-treated cells, including cell blebbing, apoptotic bodies, and nuclear fragmentation. This suggests that BCLi shifts cell fate decision in AURKAi-treated cells towards apoptosis. In conclusion, combination treatment with AURKAi and BCl2i induced p53-dependent apoptosis. Preclinical evaluation of this drug combination in PDO model demonstrated robust efficacy against melanomas with wild type p53. Since p53 mutations are relatively rare in melanoma, combination of AURKAi and BCLi presents promising therapeutic option for this deadly disease.

Keywords: Apoptosis, melanoma, patient-derived organoids, aurora kinase, bcl-2

Citation Format: Vijaya Bharti, Vivian Weiss, Chengli Shen, Sheau Chiann Chen, Ashlyn Blevins, Rebecca L. Shattuck-Brandt, Ann Richmond, Anna E. Vilgelm. Patient derived organoids demonstrate synergistic antitumor effect of senogenic and senolytic drug combination of Aurora kinase inhibitor and BCL2/xL inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1054.
Abstract 1067: PDK-1 targeting with SNS-510 in sarcoma
Authors: Musi Elgilda , Taverna Pietro , Schwartz Gary K.
Published: July 2021

Soft tissue sarcomas are heterogeneous mesenchymal neoplasms and account for 1% of all cancers in adults. Over 50 sarcomas subtypes have been classified and many have limited treatment options some of which include surgery, chemotherapy and radiation. 3-phosphoinositide dependent protein kinase 1 (PDK1) is overexpressed in many cancers, suggesting a role for PDK1 in cancer progression. In addition, PDK1 has been identified as a key modifier of acquired resistance to CDK4/6 inhibitors. In order to interrogate this in sarcoma, we elected to test SNS-510 a potent, orally bioavailable PDK1 inhibitor against a broad range of sarcoma cell lines (leiomyosarcoma, dedifferentiated liposarcoma, Ewing's sarcoma, osteosarcoma, chondrosarcoma and synovial sarcoma) as well as dedifferentiated liposarcoma cell lines with acquired resistance to ribociclib, which are also cross resistant to palbociclib. All the cell lines expressed PDK1 and, as measured by WST-8 cell proliferation assay, nearly all the cell lines were sensitive to the drug, with IC50s generally between 250 to 500nM. Leiomyosarcoma cell lines (SK-LMS, SK-UT1, SK-UT1b) were seen to be most sensitive to SNS-510 with IC50s determined to be between 50-100nM. In contrast, synovial sarcoma cell lines (SYO-1 and HSSY-II) exhibited relative resistance to SNS-510 with IC50s determined to be > 500nM. As assessed by western blot, SNS-510 exhibited a dose dependent inhibition of the PDK-1/RSK2/AKT pathway with inhibition of p-RSK2(244), p-AKT(308), and pS6(235/236). SNS-510 induced a dose-dependent G1 cell cycle arrest. This was associated with inhibition of p-RB(780) and suppression of cyclin D1 and cyclin E1. The exception to this was the synovial cell lines for which there was no inhibition of p-RB despite inhibition of p-RSK, p-AKT and p-S6. When combined with palbociclib in both the CDK4/6 sensitive (LS141 and DDLS) and CDK4/6 resistant liposarcoma cells (RES.LS141 and RES.DDLS), we observed enhanced inhibition of proliferation with a synergistic effect, as defined by the Chou-Talalay method. These results would suggest a role for PDK-1 targeting with SNS-510 in sarcoma and consideration of combination strategies, especially with CDK4/6 inhibitors in both CDK4/6 sensitive and resistant dedifferentiated liposarcoma.

Citation Format: Elgilda Musi, Pietro Taverna, Gary K. Schwartz. PDK-1 targeting with SNS-510 in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1067.
Abstract 1070: SCR6106, an oral and brain penetrant SERD demonstrated anti-tumor activities in vitro and in vivo
Authors: Zhou Feng , Liu Lei , Yang Guimei , Gu Peng , Xue Liting , Yang Wenqing , Chen Ping , Tang Renhong
Published: July 2021

Benefits of endocrine therapy in treating hormone receptor (HR)-positive (HR+) BC are well recorded in clinic. However, ~40% to 50% of HR+ patients eventually developed to endocrine resistance and disease relapsed. Fulvestrant, a selective estrogen receptor degrader (SERD) is approved to treat HR+ metastatic breast cancer in patients with disease progression after endocrine therapy. However, due to its poor bioavailability, it is given by intramuscular injection, which makes inconvenience in administration. Additionally, approximately 14% of hormone positive patients with advanced breast cancer are diagnosed with brain metastases, while, at present, there are no FDA-approved systemic therapies for the treatment of breast cancer brain metastases. Therefore, to develop a new generated oral and brain-penetrant SERD to overcome these challenges is believed to be needed. SCR6106, discovered and currently being developed by Simcere, is a novel, potent, orally delivered and non-steroidal SERD that both antagonizes and degrades ERα. SCR6106 showed high potency on inducing ERα protein degradation (DC50=0.29 nM) and anti-proliferation (IC50=0.43 nM) in MCF7 cells. In addition, SCR6106, showed high activities on ERα degradation and anti-proliferation in a panel of ER+ breast cancer cell lines. Meanwhile, ER target genes transcription were evaluated and SCR6106 exhibit strong suppressive phenotype in T-47D. SCR6106 demonstrated more potent than fulvestrant on anti-tumor growth in vivo. In MCF7 xenograft model, SCR6106 at 3 mpk QD induced 113% tumor growth inhibition (TGI), compared to fulvestrant 56.8% TGI at 250 mpk. Compared to fulvestrant, SCR6106 demonstrated stronger ERα degradation and inhibition of ER target genes transcription in tumors, which was consistent with anti-tumor activities. SCR6106 displayed good oral bioavailability in multiple pre-clinical species, which supports its oral administration in human. Significantly, SCR6106 possess a high BBB permeability, the B/P ratio is more than 4 in pre-clinical species. The exposure of SCR6106 observed in mouse brain is much higher than the IC90 value of anti-proliferation (MCF7), and SCR6106 demonstrated anti-tumor activities in a MCF-7 intracranial tumor xenograft model. In conclusion, SCR6106 is a novel, potent and oral SERD, which demonstrated anti-tumor activities in vitro and in vivo. It showed high potential ability to cross the blood-brain barrier, and could be used to treat HR+ patients with brain metastasis.

Citation Format: Feng Zhou, Lei Liu, Guimei Yang, Peng Gu, Liting Xue, Wenqing Yang, Ping Chen, Renhong Tang. SCR6106, an oral and brain penetrant SERD demonstrated anti-tumor activities in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1070.
Abstract 1084: Targeting MET and RON to overcome cetuximab resistance in colorectal cancer
Authors: Graves-Deal Ramona , Bogatcheva Galina , Damalanka Vishnu , Klampfer Lidija , Janetka James W. , Coffey Robert J. , Singh Bhuminder
Published: July 2021

Using a novel 3D culture system, we previously showed that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to cetuximab resistance in colorectal cancer (CRC) cells. CC derived from HCA-7 cells were sensitive to cetuximab, whereas similarly derived SC were resistant; we also generated cetuximab-resistant (CC-CR) upon prolonged exposure of CC to cetuximab. Both de novo and acquired modes of cetuximab resistance in SC and CC-CR, respectively, could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. Moreover, MET and RON ligands, HGF and HGFL, respectively are synthesized as inactive precursors and require cleavage by proteases (HGFA, Matriptase, and Hepsin) to be biologically active. Herein, to inhibit HGF/HGFL cleavage in cetuximab-resistant cells, we employed inhibitors of proteases of HGF/HGFL (ZFH7116 and VD2173) and were able to overcome both de novo and acquired cetuximab resistance. We are now in the process of engineering cetuximab-sensitive CC cells to overexpress HGF or HGFL to test if ligand overexpression imparts cetuximab resistance and if this resistance can be overcome by inhibition of HGF/HGFL proteases and/or by crizotinib. To understand the broader significance of these resistance mechanisms, we have identified a set of KRAS wild-type human CRC patient-derived tumor xenografts (PDXs) that show increased MET/RON phosphorylation. A comparative RNA-seq analysis showed that PDXs with high pMET/pRON levels could be segregated from PDXs with low pMET/pRON levels. Differential response of these PDXs and their organoid (PDO) derivatives for cetuximab resistance and response to crizotinib addition based on their pMET/pRON levels will be presented.

Citation Format: Ramona Graves-Deal, Galina Bogatcheva, Vishnu Damalanka, Lidija Klampfer, James W. Janetka, Robert J. Coffey, Bhuminder Singh. Targeting MET and RON to overcome cetuximab resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1084.
Abstract 1087: CXCR2 and its ligands modulate chemotherapy resistance in pancreatic ductal adenocarcinoma
Authors: Molczyk Caitlin , Prajapati Dipakkumar R. , Saxena Sugandha , Goel Paran , Varney Michelle , Singh Rakesh K.
Published: July 2021

Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest survival rates of cancers, with a five-year relative survival of 9%. The significant factor contributing to low survivorship is the intrinsic resistance to chemotherapy. In the present study, we elucidate a targetable pathway that facilitates resistance in these cells. One inflammatory pathway used in cancer progression is CXCR2, which binds chemokines CXCL 1-3 and 5-8. Our group and others have shown an association of CXCR2 with tumor progression, angiogenesis and metastasis in PDAC, and chemotherapy resistance in other cancers. In this study, we test the hypothesis that CXCR2 is integral in PDAC chemotherapy resistance. We generated gemcitabine resistant cell lines (GemR) from the parent cell lines of T3M4 (WT KRAS) and CD18/HPAF (Mutant KRAS) with 40- and 67-fold higher resistance in comparison to the parent cell lines, respectively. Using qRT-PCR and ELISA, we profiled chemokines' expression, CXCL1, 5, and 8, on parent- and GemR-cell lines. We observed a higher basal level expression of CXCL1, 5, and 8 ligands in GemR cells than the parent cell lines. Further treatment with gemcitabine, the parent cell lines had a higher increase in CXCL1 and 8 ligand expression in comparison with GemR cell lines. With further investigation of T3M4, we found a dose-dependent and time-dependent increase for CXCL1 and CXCL8. CXCL5 had a constant high expression with all doses of gemcitabine. With our initial observations of gemcitabine treatment enhancing CXCR2 ligands in PDAC cells, we evaluated the effects of the small molecule CXCR2 antagonist SCH 479833 (SCH 47) in combination with gemcitabine treatment on the parent and GemR cell lines. We used the relative IC50 and half the relative IC50 for both the gemcitabine and SCH 47 treatment doses. We observed that a lower concentration of gemcitabine in the presence of the SCH 47 was more effective than the higher concentration of gemcitabine without SCH 47 for the GemR-cell lines. Together, our observations show chemotherapy-resistant PDAC cell lines express higher CXCR2 ligand levels at baseline, with parent cell lines increasing expression with chemotherapy treatment. We also show an advantageous combination treatment of CXCR2 antagonist with lower gemcitabine concentration. Overall, these results show a promising future for CXCR2 antagonism in the treatment of resistant PDAC tumors.

Citation Format: Caitlin Molczyk, Dipakkumar R. Prajapati, Sugandha Saxena, Paran Goel, Michelle Varney, Rakesh K. Singh. CXCR2 and its ligands modulate chemotherapy resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1087.
Abstract 1118: Engineered cell line panel complemented with in vivo models to enable next-generation target therapy discovery in cancer
Authors: Zhang Zhixiang , Shi Xiaohe , Zuo Xiangyang , Gu Qingyang , Ji Qunsheng
Published: July 2021

Many cancer types are driven by specific gene aberrances, like gene fusions, mutations and copy number changes. It has led to the successful application of a number of target therapeutics, of which kinase inhibitors represent a good example. However, acquired resistance is almost inevitable at the end of any target therapy treatment, which makes it urgent a need to discover new-generation therapeutics. There are strong evidences suggesting acquired resistance should be largely attributed to certain secondary mutations, such as EGFRT790M, RETV804L and BTKC481S. At discovery stage it is critical to generate preclinical cancer models harboring these specific gene aberrances both in vitro and in vivo. Biopsy from patient with clinical resistance could be an optional starting point. But it is much more straightforward and efficient if these well-studied gene aberrance could be constructed and engineered into host cell lines directly. The transformed cell line can further be inoculated into host mice to make in vivo test feasible. Here we are reporting the establishment of a panel of engineered BaF3 cell lines for a series of cancer driver gene aberrances against EGFR, RET, ROS1, BTK and FLT3. Taking RET as example, we constructed the activating gene fusion of KIF5B-RET, along with its mutated versions, KIF5B-RETV804L and KIF5B-RETV804M. These 3 constructs were transfected into BaF3 cell line, respectively. After rounds of in vitro selection, the stable BaF3 cell line harboring KIF5B-RETWT, KIF5B-RETV804L or KIF5B-RETV804M was subjected into in vitro test of three multiple kinase inhibitors with RET inhibitory activity, including Ponatinib, Sunitinib and Sorafenib. Consistent to other reports, Ponatinib was demonstrated superior to the other two RET inhibitors when suppressing the two V804 mutated cell line growths. The in vivo growth of these cell lines have also been confirmed in house. Beyond these five target genes, we have been working on other driver genes to expand the panel scope continuously. HER2 exon20 ins, PDGFRaT674I and KITT670I models are already under development, while many others are on our radar. In summary, our ever-expanding panel of engineered cell lines complemented with in vivo models represents valuable assets to empower the discovery of next-generation target therapeutics against cancer.

Citation Format: Zhixiang Zhang, Xiaohe Shi, Xiangyang Zuo, Qingyang Gu, Qunsheng Ji. Engineered cell line panel complemented with in vivo models to enable next-generation target therapy discovery in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1118.
Abstract 1147: RNA-targeting approaches to modulate alternative RNA splicing in cancer
Authors: Leclair Nathan K. , Urbanski Laura , Brugiolo Mattia , Yurieva Marina , Graveley Brenton R. , Cheng Albert , Anczukow Olga
Published: July 2021

Alternative RNA splicing (AS) is a critical step in gene expression regulation. This differential inclusion of sequences in the mRNA is a source of functional transcriptomic diversity that allows multiple RNA isoforms to be expressed from the same gene. Dysregulation of AS is hallmark of cancer and leads to the expression of RNA isoforms that promote tumor initiation and maintenance. Understanding how tumor-associated RNA isoforms impact cancer biology is needed for the development of cancer therapies targeting AS. Here, we describe two approaches to modulate AS in cancer cells. First, we utilize CRISPR-Artificial Splicing Factors (CASFx), splicing factor domains fused to catalytically inactive Cas13 proteins, to direct splicing activity to a specific site using a guide RNA (gRNA). We demonstrate that this approach can be used to promote either exon inclusion or skipping in mRNA from several cancer-associated genes (e.g., HRAS, EP400, TRA2β, SRSF3) depending on the gRNA binding location and the splicing-factor domain fusion. Second, we develop antisense oligonucleotides (ASOs) that target a non-coding poison exon in the oncogenic splicing factor TRA2β. This exon functions as a regulatory “poison exon”, which induces RNA degradation when included, and is differentially spliced in multiple tumor types compared to their corresponding adjacent normal tissue. TRA2β-targeting ASOs promoting poison exon inclusion cause decreased TRA2β protein expression, increased cell death, and decreased proliferation across multiple cancer cell lines. Additionally, compared to cancer cells, normal cell lines were less affected by TRA2β-targeting ASO toxicity, demonstrating a therapeutic window for using this technology to specifically target cancer cells.

Citation Format: Nathan K. Leclair, Laura Urbanski, Mattia Brugiolo, Marina Yurieva, Brenton R. Graveley, Albert Cheng, Olga Anczukow. RNA-targeting approaches to modulate alternative RNA splicing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1147.
Abstract 1166: Structurally defined glycosaminoglycan mimetics exhibit preference for proteins of the growth factor family
Authors: O'Hara Connor P. , Sankaranarayanan Nehru V. , Boothello Rio S. , Patel Bhaumik B. , Desai Umesh R.
Published: July 2021

Heparan sulfate (HS), a glycosaminoglycans of considerable structural diversity, has been implicated in a wide-variety of complex roles including regulation of cell signaling. Unfortunately, homogeneous HS is a dream that will likely be unfulfilled this century. To combat this, our lab has synthesized an array of non-saccharide glycosaminoglycan mimetics (NSGMs) that act as structural and functional mimetics of HS. A key advantage of our NSGMs is homogeneity, which enables assessing selectivity for recognition of target proteins with a high degree of confidence. A second advantage is the quantitative measurement of the affinity of these interactions, which affords a detailed understanding of the forces governing recognition of different proteins. Previously, we have shown a lead NSGM named G2.2 as a potent and selective inhibitor of colorectal cancer stem cells (CSCs) through phenotypic in-vitro and in-vivo assays. To improve upon the drug-like properties of G2.2, cholesterol-modified analogs (G2C, G5C, G8C) were designed to enhance potency and selectivity for protein target(s). Although the hypothesis is that G2.2 and its lipidic analogs target a ligand and/or a receptor belonging to CSC-relevant growth factors, the preferred target, if any, remains to be identified. More importantly, the relative order of preference in-vitro and its use in predicted in-vivo activity remains to be determined. In this work, we have performed a comprehensive biophysical screening of a panel of potential growth factor targets using biophysical methods to determine binding affinities, followed by quantitative measurement of the relative forces of interactions involved in target recognition. Our studies are complemented by computational experiments that provide a foundation for thermodynamic studies. Analysis of our results indicate that these NSGMs preferentially recognize certain growth factor proteins, suggesting that appropriately designed HS mimetics can be tailored to modulate a particular tyrosine kinase receptor pathway. More importantly, our results convey the principle that despite the presence of multiple sulfate groups, HS mimetics can be designed to exhibit selectivity for certain growth factor proteins. In fact, the sulfate groups on our molecules may actually contribute to the selectivity and potency of recognition, two central principles for the development of drugs.

Citation Format: Connor P. O'Hara, Nehru V. Sankaranarayanan, Rio S. Boothello, Bhaumik B. Patel, Umesh R. Desai. Structurally defined glycosaminoglycan mimetics exhibit preference for proteins of the growth factor family [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1166.
Abstract 1187: Optimizing gemcitabine in metastatic TNBC by rational PI3K/AKT combination
Authors: Merrill Nathan M. , Vandecan Nathalie M. , Apfel Athena M. , Ulintz Peter J. , Bao Liwei , Cheng Xu , Morikawa Aki , Merajver Sofia D. , Soellner Matthew B.
Published: July 2021

Metastatic triple-negative breast cancer (TNBC) has a median survival of 13 months, and only 5 months if brain metastases are present. Advances in immunotherapy are promising, but only add weeks to a few months of progression-free survival to this guarded prognosis. Thus, there is a large unmet need for innovations that improve patient outcomes in the metastatic setting when patients are refractory to first-line treatment. Recently, we published an innovative strategy to identify multi-omic molecular correlates of drug response in TNBC. From this work, we now apply the strategy to 3D cell culture models and patient derived xenografts (PDXs) that are more representative of in vivo drug response in real patients. In brief, we have tested the dose response for a panel of 387 investigational drugs and plotted this readout against molecular characterization in 35 TNBC cell lines and PDXs. Using linear regression, we have identified statistically significant positively and negatively correlating DNA, RNA, and protein predictors of drug efficacy. From this expanded multi-omic set, we have identified a multi-omic signature for sensitivity to gemcitabine, a drug widely used in TNBC, but at present, without any a priori knowledge of who is likely to benefit. Moreover, we noted a significant correlation of response with MERIT40, an AKT substrate. MERIT40 is critical for localization of BRCA1 to facilitate DNA damage repair. MERIT40 promotes resolution of DNA damage caused by chemotherapy, and thus, based on our published work and supporting literature, PI3K/AKT inhibition is a rational synergistic partner with gemcitabine. Moreover, further supporting our hypothesis, preliminary results indicate Chou-Talalay synergy at therapeutically relevant doses when gemcitabine is combined with PI3K/AKT inhibition. In future studies, we will evaluate the ability of the gemcitabine biomarker to predict response, and the efficacy of the prioritized combinations using ex vivo patient models of metastatic breast cancer established in our laboratory.

Citation Format: Nathan M. Merrill, Nathalie M. Vandecan, Athena M. Apfel, Peter J. Ulintz, Liwei Bao, Xu Cheng, Aki Morikawa, Sofia D. Merajver, Matthew B. Soellner. Optimizing gemcitabine in metastatic TNBC by rational PI3K/AKT combination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1187.
Abstract 1189: Association of RAS pathway mutations with lower CD8+ T cell infiltration and 2-year survival rate in Stage-III colorectal adenocarcinoma patients
Authors: Jiang Zhipeng , Zhao Feilong , Chen Hui , Zhao Xiaochen , Bai Yuezong
Published: July 2021

Background: The oncogenic pathways, including WNT, TP53, RAS, PI3K and TGFβ, were associated with tumor progression in colorectal adenocarcinomas (CRC). However, a majority of CRC patients have mutations from multiple pathways simultaneously, which makes it difficult to identify the key pathway influencing prognosis of CRC. Herein, we aimed to evaluate the novel dominant pathway-typing method in predicting the prognosis of Stage-III CRC.

Methods: Clinicopathologic and molecular variation data of Stage-III CRC patients (n=152) were obtained from The Cancer Genome Atlas database (TCGA). Immune cell infiltration level of the tumor was calculated by xCell (https://xcell.ucsf.edu/). Oncogenic pathway genes were classified according to a TCGA study (PMID: 29625050). In each patient, the dominant pathway-typing were based on which pathway the genes with the highest variant-allele-frequency (VAF) belongs to.

Results: In all, 124/152 (82%) patients had mutation on at least one of the oncogenic pathway genes and 120/124 (97%) patients harbored mutations from two or more pathways simultaneously. According to their dominant pathway-typing method, the patients were classified into five groups: WNT (n=45), RAS (n=26), TP53 (n=36), PI3K (n=8) and TGFβ (n=9) groups. There was no significant difference in OS among the five groups. However, the two-year survival rate of RAS group was significantly lower than that of WNT (p=0.042) and TP53 (p=0.035) groups. Further, we found that the infiltration of CD8+ T cell was significantly lower in RAS group compared to WNT group (with median infiltration score: 0.005 vs 0.013, p=0.024). Importantly, we found that among the RAS group without CD8+ T-cell infiltration, 63% (5/8) patients were died within two years after diagnosis. While the two-year death rate of WNT and TP53 groups without CD8+ T cell infiltration were zero (0/6) and 14% (1/7). And the two-year death rate of RAS, WNT and TP53 groups with CD8+ T cell infiltration were 17% (1/6), 11% (3/27) and 8% (1/13), respectively.

Conclusion: RAS pathway mutation may be associated with low CD8+ T cell infiltration and promote the two-year death rate of stage-III CRC patients using dominant pathway-typing method. And further larger cohorts are needed to validate these findings and the underlying mechanisms.

Citation Format: Zhipeng Jiang, Feilong Zhao, Hui Chen, Xiaochen Zhao, Yuezong Bai. Association of RAS pathway mutations with lower CD8+ T cell infiltration and 2-year survival rate in Stage-III colorectal adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1189.
Abstract 1194: MEK inhibition in combination with PI3K/AKT/MTOR is a promising therapeutic in basal bladder cancer
Authors: Apfel Athena M. , Merrill Nathan M. , Vandecan Nathalie M. , Bao Liwei , Cheng Xu , Lopez-Barcons Lluis A. , Day Kathleen C. , Palmbos Phillip L. , Day Mark L. , Udager Aaron M. , Soellner Matthew B. , Merajver Sofia D.
Published: July 2021

There are diverse resources available for data relevant to the drug screening of bladder cancer cell lines in 2D culture, such as the Cancer Cell Line Encyclopedia (CCLE) and Genomics of Drug Sensitivity in Cancer (GDSC) databases; however, 3D culture is more representative of in vivo response in patients. Moreover, drug screens in 3D reveal differential drug response and expression of genes and proteins that indicate the potential of more effective selection of drugs for specific patients: the basic tenet of personalized oncology. Therefore, we screened bladder cancer cell lines in 3D culture using a library of curated small-molecule inhibitors to identify compounds and classes of compounds with efficacy in bladder cancer. Genomic and transcriptomic data for these bladder cancer cell lines were used to correlate genomic molecular parameters with drug response with the aim of discerning biomarkers of response or to suggest plausible drug combinations. This allowed us to identify groups of tumors that are vulnerable to specific drugs or classes of drugs. We found that MEK inhibitors were a promising target among basal bladder cancers, an RNA-based molecular subtype of bladder cancer, especially aggressive and exhibiting poor prognosis. Furthermore, trametinib was found to be a “very active” MEK inhibitor in basal cancers, classified using drug sensitivity 3 (DSS3) scoring. PI3K/AKT/MTOR inhibitors target a parallel signaling pathway to MEK inhibition; therefore, we tested PI3K/AKT/MTOR combinations with trametinib in basal bladder cancer to query for synergistic properties. Additionally, we tested PI3K/AKT/MTOR combinations with trametinib using ex vivo organoids of bladder cancer patient samples collected directly at operation. Taken together, MEK inhibition is a promising therapeutic strategy in the basal subtype of bladder cancer, particularly in combination with parallel pathway inhibition. Moving forward, we plan to test PI3K/AKT/MTOR combinations with trametinib in trametinib-resistant basal bladder cancer cell lines to investigate how this affects signaling and re-sensitization to trametinib.

Citation Format: Athena M. Apfel, Nathan M. Merrill, Nathalie M. Vandecan, Liwei Bao, Xu Cheng, Lluis A. Lopez-Barcons, Kathleen C. Day, Phillip L. Palmbos, Mark L. Day, Aaron M. Udager, Matthew B. Soellner, Sofia D. Merajver. MEK inhibition in combination with PI3K/AKT/MTOR is a promising therapeutic in basal bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1194.
Abstract 1196: BALF liquid biopsy provides a novel platform for speedy and accurate EGFR mutation testing in advanced NSCLC patients
Authors: Kim In Ae , Hur Jae Young , Kim Hee Joung , Kim Wan Seop , Lee Kye Young
Published: July 2021

Background

EGFR mutation testing is an essential step for the therapeutic decision in newly diagnosed advanced NSCLC patients and usually performed by using DNA extracted from biopsied tumor tissue with a turnaround time(TAT) of 2-3 weeks. Plasma liquid biopsy using ctDNA is minimally invasive and less time-consuming, but shows limited usefulness due to low sensitivity. We conducted a prospective study to demonstrate clinical usefulness of bronchoalveolar lavage fluid (BALF) liquid biopsy using extracellular vesicle(EV)-derived DNA in advanced NSCLC patients by comparing the sensitivity and TAT with conventional tissue biopsy and plasma liquid biopsy

Methods

The analysis set for liquid EGFR genotyping is 224 BALF samples and 110 plasma samples obtained from newly diagnosed 224 advanced non-squamous NSCLC patients. EGFR genotyping was done through peptide nucleic acid(PNA)-mediated real-time PCR after extracting BALF EV DNA and plasma cfDNA separately. The sensitivity, specificity, and concordance rate of BALF liquid biopsy were calculated in comparison to matched tissue genotyping and plasma liquid biopsy. In addition, TAT was compared between BALF liquid biopsy and tissue genotyping.

Results

Tissue genotyping revealed 93 EGFR mutant cases (41.5%) and 131 wild-type cases (58.5%). The sensitivity, specificity, positive predictive value, negative predictive value, and concordance rate of BALF liquid biopsy to tissue genotyping were 97.8%, 96.9%, 96.8%, 98.4%, and 97.7%, respectively. BALF liquid biopsy showed almost identical performance with standard tissue genotyping, while only 5 cases were mismatched each other. On the other hand, plasma liquid biopsy using cfDNA (n=110) revealed 48.5% sensitivity, 86.3% specificity, and 63.6% concordance rate in comparison to tissue genotyping. The mean turnaround times of BALF liquid biopsy was significantly shortened to 2.6±2.0 days in contrast to 13.9±12.4 days of tissue genotyping. (p<0.001).

Conclusion

BALF liquid biopsy can be developed as a novel platform for EGFR mutation testing in advanced NSCLC with almost identical performance with conventional tissue genotyping and significantly shorten TAT to 1-2 days.

Citation Format: In Ae Kim, Jae Young Hur, Hee Joung Kim, Wan Seop Kim, Kye Young Lee. BALF liquid biopsy provides a novel platform for speedy and accurate EGFR mutation testing in advanced NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1196.
Abstract 1204: SO-N102, a novel CLDN18.2-targeting antibody-drug conjugate with strong anti-tumor effect in various solid tumors expressing low target levels
Authors: Sadilkova Lenka Kyrych , Valentova Iva , Hoskova Simona , Vopalensky Pavel , Bammert Lukas , Beerli Roger , Moebius Ulrich , Spisek Radek
Published: July 2021

Claudin (CLDN) 18.2, a member of a large family of transmembrane proteins with distinct functions, has been shown to have a high prevalence, predominantly in gastric and pancreatic cancer. In addition, ectopic expression of CLDN18.2 was described for other cancer types including ovarian, lung, liver and colon, whereas healthy tissue expression is restricted to the stomach. SO-N102 represents a CLDN18.2 targeting antibody-drug conjugate based on a novel proprietary highly specific monoclonal antibody conjugated to a derivative of PNU-159682 using SMACTM technology for the therapy of patients with various CLDN18.2-positive solid tumors, mainly of gastric and pancreatic origin. The CLDN18.2 protein sequence is highly conserved in mammalians with 100% identity in the targeted extracellular loop among rodents, cynomolgus monkey and human. SO-N102 showed excellent specificity for CLDN18.2, strong binding to the target followed by efficient tumor cell killing. Preferential binding to selected patient-derived tumor tissues was observed ex vivo when compared to healthy stomach tissues from mice and cynomolgus monkey. Single-agent therapeutic activity of SO-N102 was demonstrated in 10 patient-derived mouse xenografts models (gastric, pancreatic, liver, colon and lung adenocarcinomas). Complete responses were observed in all models, independent of CLDN18.2 expression levels, ranging from low (IHC1+) to high (IHC3+), with minimum efficacious doses between 0.2 mg/kg and 0.6 mg/kg. An acceptable tolerability profile was observed in preliminary toxicity studies at 10 mg/kg (mouse), 6 mg/kg (rat) and 1 mg/kg (cynomolgus monkey). SO-N102 demonstrated favorable pharmacokinetic properties with half-lives in the range of 8 days and 13 days in cynomolgus monkey and rat, respectively. Stability of SO-N102 without any significant loss of the payload was demonstrated in vitro and in animals. Further toxicology studies in rats and cynomolgus monkeys were initiated, paralleled by process development and manufacturing activities with the plan to initiate the first clinical study with SO-N102 in patients in the first half of 2022.

Citation Format: Lenka Kyrych Sadilkova, Iva Valentova, Simona Hoskova, Pavel Vopalensky, Lukas Bammert, Roger Beerli, Ulrich Moebius, Radek Spisek. SO-N102, a novel CLDN18.2-targeting antibody-drug conjugate with strong anti-tumor effect in various solid tumors expressing low target levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1204.