bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1009: Combination Niraparib and Metformin synergistically inhibits endometrial cancer cell growth and metastasis
Authors: Gan Qiyu , Cheng Jingxin , Chen Rui , Bie Yachun , Wang Guozeng , Chang Linlin
Published: July 2021

Backgroud Endometrial Carcinoma (EC) is the most common gynecological malignancy among women. Even with a favorable prognosis, cure is still elusive with recurrence, chemotherapy resistance and poor outcome. Poly ADP-ribose polymerase inhibitors (PARPi) have been emerged as promising cancer therapeutics, especially for tumors with deficient homologous recombination (HR) repair. Niraparib for example is well established for ovarian cancer. Metformin, an antidiabetic drug, has been reported to be a new adjunctive strategy for different cancer types, including endometrial cancer. While the effect of Niraparib for EC as monotherapy or in combination with Metformin is still unknown.

Method Here we used two types of endometrial cancer cell lines Ishikawa and HEC-1B to detect the effect of Niraparib as monotherapy or in combination with Metformin on cell proliferation, apoptosis, invasion and metastasis. The 50% Inhibitory concentration (IC50) was assessed, and transwell, Annexin V staining, clonogenic assay were applied. Also, western blotting was performed to determine the expression of proteins involved, such as Bax, Caspase-3, Bcl-2, p-AKT, RAD51, γ-H2AX and so on.

Results Our study demonstrated that Niraparib inhibited endometrial cell growth in both Ishikawa and HEC-1B cell lines with IC50 0.024umol/L and 0.127umol/L separately. The invasion and metastasis were also suppressed. Annexin V staining showed that Niraparib could significantly promoted apoptosis, which was largely enhanced by Metformin combination therapy (p<0.01). Accordingly, the apoptosis-related proteins Bax, Caspase-3, and DNA damage-related protein RAD51, γ-H2AX were increased, while proliferation-related protein p-AKT, anti-apoptosis-related protein Bcl-2, invasion and metastasis-related protein E-cadherin were decreased, as confirmed by western blotting.

Conclusion Niraparib shows the anticancer effect on endometrial cancer cell through inhibiting the cell viability, metastasis and inducing apoptosis, which is significantly enhanced by the combination with Metformin. Our results provide preclinical evidence that the combination of these well-establised drugs may warrant further clinical investigation for endometrial cancer treatment.

Citation Format: Qiyu Gan, Jingxin Cheng, Rui Chen, Yachun Bie, Guozeng Wang, Linlin Chang. Combination Niraparib and Metformin synergistically inhibits endometrial cancer cell growth and metastasis [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 1009.
Abstract 1015: mTOR inhibitor induces non-apoptotic, autophagy dependent cell death (ADCD) in TP53 mutant HNSCC
Authors: Alam Md Maksudul , Fermin Janmaris Marin , Spiller Patrick T. , Dorr Channing , Rong Xiaohua , Medlin Tara-Moore , Khandelwal Alok R. , Nathan Cherie-Ann
Published: July 2021

TP53 is the most frequently mutated gene in Head and Neck Squamous Cell Carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis. A phase II multi-institutional clinical trial, determined that, adjuvant therapy with an mTOR inhibitor significantly increased 2-year progression free survival (PFS) in patients with p53 mutant tumors. Previous studies have shown that mutant p53 causes sustained activation of PI3K/AKT/mTOR pathway in cancer. We investigated the mechanism by which the mTOR inhibitor CCI-779 inhibits cell growth of TP53 mutant HNSCC in vitro. HPV-negative HNSCC cell lines harboring TP53 mutations at different positions were treated with CCI-779.CCI-779 significantly reduced cell viability and colony forming ability of all the cell lines tested. CCI-779 did not induce apoptosis or cell cycle arrest in these cells. However, the reduction in cell viability was associated with increase in autophagy as demonstrated by increased levels of LC3-II. The autophagy inhibitors, 3-methyl adenine (3-MA) and ULK-101 restored the cell viability by inhibiting CCI-779 induced autophagy. Therefore, we postulate that CCI-779 induces nonapototic autophagy dependent cell death (ADCD) in HPV-negative TP53 mutant HNSCC cell lines. CCI-779 induced autophagy is accompanied by increased level of ULK1 and Beclin1 as well as increased phosphorylation of ULK1 at Serine 555. However, ULK101 (ULK1 inhibitor) and 3-MA (PI3K inhibitor) when combined with CCI-779 reduced the level of both total and phosphorylated levels of ULK1, which resulted in inhibition of autophagy and improved cell viability. However, combination of CCI-779 and bafilomycin A1 treatment showed increased level of LC3-II, ULK1 and phospho-ULK1 (S555) with concomitant decrease in cell viability. Our data suggests that rapalog induces ADCD by facilitating autophagy initiation through the stabilization and activation of ULK1. Moreover our data demonstrated that CCI-779 mediated activation of ULK1 is associated with down regulation of mTORC1 but not mTORC2 signaling. This is a novel mechanism of how rapalogues induce ADCD through the stabilization/activation of ULK1 in HPV-negative TP53 mutant HNSCC cells. Together these data highlight the importance of how mTORi can be used to accelerate tumor cell death in HPV-negative TP53 mutant HNSCC.

Citation Format: Md Maksudul Alam, Janmaris Marin Fermin, Patrick T. Spiller, Channing Dorr, Xiaohua Rong, Tara-Moore Medlin, Alok R. Khandelwal, Cherie-Ann Nathan. mTOR inhibitor induces non-apoptotic, autophagy dependent cell death (ADCD) in TP53 mutant HNSCC [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 1015.
Abstract 1056: Potent in vivo anti-tumor activity of D-1553 as a single agent and in combination with targeted therapeutics in a broad spectrum of patient-derived xenograft tumor models with KRas G12C mutation
Authors: Shi Zhe , Weng Jifang , Fan Xiaochong , Zhu Qingqing , Robb Emily , Moriarty Alyssa , Wick Michael , Jiang Yueheng , Zhang Ling , Dai Xing , Wang Yaolin
Published: July 2021

Ras protein plays a critical role in cell growth and proliferation by transmitting signal from cell surface receptor tyrosine kinase to downstream cellular proteins such as RAF and MEK. KRas is one of the most frequently mutated oncogenes, with mutations at residues G12, G13, and Q61, found in lung, colon, pancreatic and other solid malignancies. We have developed an orally bioavailable, covalent inhibitor of KRas G12C, D-1553, with potent in vitro biochemical, cellular activity and in vivo efficacy. Here we report the efficacy of D-1553 in a large panel of patient-derived xenograft (PDX) tumor models with KRas G12C mutation. In the lung cancer PDX models, D-1553 exhibited tumor growth inhibition (TGI) from 43.6% to 124.3%, with 4 out of 8 models showing tumor regression. In the colorectal cancer PDX models, the range of TGI was from 60.9% to 105.7%, with 3 out of 9 models showing regression. Combination treatment of D-1553 with chemotherapy and targeted agents demonstrated enhanced anti-tumor activity resulting in more tumor regression compared to single agent treatment. These observations from PDX models support the combination treatment as a key to increased overall response rate to KRas G12C inhibitor in clinical trial. D-1553 is currently in a Phase 1/2 clinical trial for patients with advanced solid tumors harboring KRas G12C mutation (NCT04585035).

Citation Format: Zhe Shi, Jifang Weng, Xiaochong Fan, Qingqing Zhu, Emily Robb, Alyssa Moriarty, Michael Wick, Yueheng Jiang, Ling Zhang, Xing Dai, Yaolin Wang. Potent in vivo anti-tumor activity of D-1553 as a single agent and in combination with targeted therapeutics in a broad spectrum of patient-derived xenograft tumor models with KRas G12C mutation [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 1056.
Abstract 1083: A novel role of SRC in mediating bypass resistance to MEK inhibition in colorectal cancer stem cells
Authors: Yang Mingli , Davis Thomas B. Davis B. , Nebozhyn Michael V. , Loboda Andrey , Wang Heiman , Schell Michael J. , Pflieger Lance , Thota Ramya , Pledger W. Jack , Yeatman Timothy J.
Published: July 2021

Colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors (KRASi/BRAFi/MEKi/ERKi), has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms including intrinsic and adaptive resistance. Here we report a central role of SRC in mediating intrinsic and adaptive “bypass”-resistance to MEK inhibition in cancer stem cells (CSCs). The SRC oncogene is a well-studied non-receptor tyrosine kinase that can regulate multiple signaling pathways involved in the control of cell proliferation, survival, differentiation, adhesion, invasion and motility. Previously, our laboratory was the first to document that human CRC truncating mutations in the negative regulatory domain of SRC result in SRC activation (Nat Genet 1999). Now our integrated and expansive gene expression signature analyses in >2000 human CRCs reveal that MEKi bypass-resistance is strikingly-correlated with SRC activation, which is associated clinically with regional metastasis, disease recurrence and poor overall survival, and associated biologically with EMT and “stemness”. In support of this, SRC-associated MEKi resistance is strongly related to the CMS4 subtype that has a mesenchymal CSC phenotype. The finding is further supported by in vitro analyses demonstrating that CSCs vs. non-CSCs display greater levels of MEKi-resistance, which is attenuated by a SRC inhibitor. These results support a novel notion that SRC may serve as a common targetable node in MEKi resistance mechanisms, permitting effective cancer stem cell targeting. These pre-clinical data, based on strikingly-correlated pathway analyses of thousands of human tumors, support the “fast-track” development of a biomarker-driven (MEKi + SRCi) drug combination targeting problematic SRC-mediated, mesenchymal CSCs.

Citation Format: Mingli Yang, Thomas B. Davis B. Davis, Michael V. Nebozhyn, Andrey Loboda, Heiman Wang, Michael J. Schell, Lance Pflieger, Ramya Thota, W. Jack Pledger, Timothy J. Yeatman. A novel role of SRC in mediating bypass resistance to MEK inhibition in colorectal cancer stem 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 1083.
Abstract 1109: A novel strategy to cope with osimertinib resistance in non-small cell lung cancer by treatment with a PIM kinase inhibitor in combination with a MERTK-selective kinase inhibitor
Authors: Yan Dan , Tan Zikang , Wang Xiaodong , Frye Stephen V. , Earp H. Shelton , DeRyckere Deborah , Graham Douglas K.
Published: July 2021

Osimertinib is currently the preferred treatment for EGFR-mutated non-small cell lung cancer (NSCLC) patients due to its superior therapeutic efficacy and prolonged overall survival compared to earlier generation EGFR tyrosine kinase inhibitors, but durable responses to osimertinib treatment are rare due to acquired drug resistance. Thus, there is an urgent need for novel strategies to treat osimertinib-resistant NSCLC. Recently, we found that treatment with MRX-2843, a novel MERTK-selective kinase inhibitor currently in Phase I clinical trials, resulted in dose-dependent inhibition of cell expansion and colony formation in an osimertinib-resistant (osiR) H4006 derivative cell line. An unbiased screen of 378 kinase inhibitors was carried out to identify compounds that synergized with MRX-2843 to inhibit expansion of an osiR derivative of the EGFR-mutated H4011 cell line. Treatment with 1µM PIM kinase inhibitor SGI-1776 or 100nM MRX-2843 alone reduced cell density by 5±3% and 44±7%, respectively, while treatment with MRX-2843 and SGI-1776 combined mediated an 82±0.4% decrease. Synergy was also observed in H4006 osiR and H1650 osiR derivative cell lines. Furthermore, treatment with PIM447, a structurally distinct PIM kinase inhibitor, and MRX-2843 decreased cell expansion more effectively than either agent alone. Mechanistically, treatment with a PIM kinase inhibitor in combination with MRX-2843 decreased downstream PI3K-AKT and MAPK-ERK signaling more effectively than single agents. Additionally, combined treatment with MRX-2843 and SGI-1776 prevented colony formation, while single agents had limited effect. In all, these data indicate that combining MRX-2843 and a PIM TKI may control osimertinib resistant tumor growth, providing a potential treatment strategy for osimertinib resistant EGFR-mutated NSCLC patients for whom the choices are still limited.

Citation Format: Dan Yan, Zikang Tan, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, III, Deborah DeRyckere, Douglas K. Graham. A novel strategy to cope with osimertinib resistance in non-small cell lung cancer by treatment with a PIM kinase inhibitor in combination with a MERTK-selective kinase 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 1109.
Abstract 115: Neutrophil extracellular traps express PD-L1 which promotes T cell exhaustion in the tumor microenvironment
Authors: Kaltenmeier Christof , Yazdani Hamza , Tohme Samer
Published: July 2021

Neutrophil extracellular traps (NETs) have recently been implicated in the growth of tumors, however their impact on immune cells within the tumor microenvironment (TME) remain incompletely understood. We hypothesize that NETs can directly interact with infiltrating T cells to promote an immunosuppressive TME. We performed liver Ischemia/Reperfusion (I/R) in an established colorectal cancer metastasis model to induce NET formation in the TME. In this NET-rich TME we discovered elevated levels of phenotypically and functionally exhausted T cells. We demonstrated that the majority of CD4 and CD8 T cells are not effector cells, but rather express multiple inhibitory receptors and are highly dysfunctional, with reduced cytokine production and proliferative capacity. This effect was limited to the T cells present the the NET-rich TME in comparison to T cells isolated from the spleen. In vitro, NETs contained the immunosuppressive ligand PD-L1 responsible for T cell exhaustion; an effect abrogated by using PD-L1 KO NETs. This exhausted state was directly linked to mitochondrial dysfunction in the T cells. Furthermore, NETs were targeted in vivo by treating mice with DNAse or anti-PD-L1 at time of I/R. Treatment caused decreased metastatic burden, decreased NET formation and higher levels of functioning non-exhausted T cells. Conversely, injecting NETs in tumors led to acceleration of tumor growth and T cell exhaustion in the TME. In addition, we measured sPDL-1 and MPO-DNA, a NET marker, in the serum of patients undergoing surgery for colorectal liver metastases resection. High levels of both markers were significantly associated with decreased disease free survival. Neutrophils isolated from patients after surgery were primed to form NETs that caused a similar exhaustion and dysfunction of human CD4 and CD8 T cells. Our data thus reveal that NETs have the capability of suppressing T cell responses and promoting tumor growth. This mechanism of T cell suppression by NETs represents a viable target for sustaining immune competence within the TME.

Citation Format: Christof Kaltenmeier, Hamza Yazdani, Samer Tohme. Neutrophil extracellular traps express PD-L1 which promotes T cell exhaustion in the tumor microenvironment [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 115.
Abstract 117: Inhibition of Hedgehog signaling alters fibroblast composition in pancreatic cancer
Authors: Steele Nina G. , Biffi Giulia , Kemp Samantha , Zhang Yaqing , Drouillard Donovan , Syu LiJyun , Hao Yuan , Oni Tobiloba , Brosnan Erin , Elyada Ela , Doshi Abhishek , Hansma Christa , Espinoza Carlos , Abbas Ahmed , The Stephanie , Irizarry-Negron Valerie , Halbrook Christopher , Franks Nicole , Hoffman Megan , Carpenter Eileen , Nwosu Zeribe , Park Youngkyu , Crawford Howard , Lyssiotis Costas , Frankel Timothy , Rao Arvind , Bednar Filip , Dlugosz Andrzej , Preall Jonathan , Tuveson David , Allen Benjamin , Pasca di Magliano Marina
Published: July 2021

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog (HH) pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of HH signaling in PDAC have been contradictory, with HH signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how HH pathway inhibition reprograms the PDAC microenvironment.

Experimental Design: We used a combination of pharmacologic inhibition, gain- and loss- of-function genetic experiments, CyTOF, and single cell RNA-sequencing to study the roles of HH signaling in PDAC.

Results: We find that HH signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAFs) compared to inflammatory CAFs (iCAFs). SHH overexpression promotes tumor growth, while HH pathway inhibition with the Smoothened antagonist LDE225 impairs tumor growth. Further, HH pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in Tregs, consistent with increased immune suppression.

Conclusions: HH pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.

Citation Format: Nina G. Steele, Giulia Biffi, Samantha Kemp, Yaqing Zhang, Donovan Drouillard, LiJyun Syu, Yuan Hao, Tobiloba Oni, Erin Brosnan, Ela Elyada, Abhishek Doshi, Christa Hansma, Carlos Espinoza, Ahmed Abbas, Stephanie The, Valerie Irizarry-Negron, Christopher Halbrook, Nicole Franks, Megan Hoffman, Eileen Carpenter, Zeribe Nwosu, Youngkyu Park, Howard Crawford, Costas Lyssiotis, Timothy Frankel, Arvind Rao, Filip Bednar, Andrzej Dlugosz, Jonathan Preall, David Tuveson, Benjamin Allen, Marina Pasca di Magliano. Inhibition of Hedgehog signaling alters fibroblast composition in pancreatic 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 117.
Abstract 1282: IND-enabling characterization of the selective GPER agonist, LNS8801
Authors: Natale Chris , Garyantes Tina
Published: July 2021

LNS8801 is the first-in-class, small molecule, orally bioavailable, agonist of GPER being developed for the treatment of patients with advanced cancer. LNS8801 is highly selective for GPER over the classical nuclear hormone estrogen receptors α and β, and it was also highly selective for GPER in Eurofins' Safety47 panel. LNS8801 has very low aqueous solubility and is highly protein bound. Pharmacokinetic studies demonstrate that LNS8801 has high clearance and moderate, solubility-dependent, oral bioavailability. The rank ordering of metabolic liability in hepatocytes is dog<human<mouse<rat, which is consistent with the observed clearance rates in these species. LNS8801 metabolism, and thus clearance, is slower in dogs. Oral bioavailability is ~80% and dose proportional in human formulations tested in dogs. LNS8801 is primarily metabolized by oxidation followed by phase II conjugation. In vitro metabolism studies indicate that LNS8801 is a potential substrate for several cytochrome P450 enzymes and thus has a low potential for participating in drug-drug interactions (DDIs). Given that maximal efficacy is expected at low exposures (Cmax of ≤5 nM) and that LNS8801 is highly protein bound, it is unlikely that these effects will cause adverse DDIs in humans. In the pivotal 28-day toxicity studies, the no-observed-adverse-effect level (NOAEL) was >1000 mg/kg/day (the maximum feasible dose) in the rat and 30 mg/kg/day in the dog. Exposure was higher in dogs than in rats due to considerable accumulation. In the dog, mortality at doses that gave very high exposures was attributed to myelopoietic hypocellularity of the bone marrow and subsequent lymphoid depletion in associated organs. The primary target organs were identified as bone marrow, lymphoid tissues, and reproductive organs. Exposure at the canine NOAEL was more than 24,000 times the predicted maximally efficacious exposure in humans, and thus LNS8801 is anticipated to be very safe.

Citation Format: Chris Natale, Tina Garyantes. IND-enabling characterization of the selective GPER agonist, LNS8801 [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 1282.
Abstract 1401: MYC mediates retinoic acid resistance by suppressing cellular retinoic acid-binding protein (CRABP2) transcription in HER2-enriched breast cancers
Authors: Choi Won-Shik , Liu Rong-Zong , Godbout Roseline
Published: July 2021

Introduction: HER2-positive (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. The development of a targeted drug, trastuzumab, has greatly improved clinical outcome for HER2+ patients. However, intrinsic and acquired resistance to trastuzumab are common. Clearly, additional therapeutic interventions are required for the clinical management of HER2-enriched breast cancers. Retinoic acid (RA) is a vital signaling molecule that regulates multiple biological processes, including cell proliferation, differentiation, and death. RA has been successfully used in the treatment of acute promyelocytic leukemia. RA also holds promise for the treatment of solid cancers like breast cancer. RA action is mediated through transcription factor RARα and the RA binding protein CRABP2, which deliver RA to RARα in the nucleus. The HER2 gene (ERBB2) is frequently co-amplified with the gene encoding RARα, a key determinant of RA sensitivity. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. MYC is an oncogene that inhibits RARα activity in leukemia cells. Importantly, MYC is preferentially amplified and overexpressed in HER2+ breast cancers. My research aims to elucidate the mechanism underlying RA resistance in HER2+ breast cancers, with a special focus on the role of MYC.

Hypothesis: MYC attenuates RA action by inhibiting the CRABP2-RARα pathway.

Results: RNA levels of MYC are negatively correlated with CRABP2 RNA levels. Depletion of MYC upregulates CRABP2 at both the RNA and protein levels. Furthermore, we found that MYC binds to the CRABP2 promoter region suggesting that MYC may directly suppress CRABP2 gene transcription activity. Our results show that ectopic expression of MYC inhibits, whereas depletion of MYC activates RAR activity. Consistently, ectopic expression of MYC increases RA resistance, whereas depletion of MYC sensitizes cells to RA treatment. When CRABP2 is depleted along with MYC-knockdown, cell proliferation is rescued, suggesting that MYC mediates RA resistance, at least partially through downregulation of CRABP2. We also found that RA treatment enhances trastuzumab responsiveness in HER2+ breast cancer cells.

Significance: This study sheds light on the role and mechanism of MYC in governing RA resistance in HER2+ breast cancer cells. Our results support the use of RA and trastuzumab for the treatment of subsets of patients with HER2+/low MYC breast cancers.

Citation Format: Won-Shik Choi, Rong-Zong Liu, Roseline Godbout. MYC mediates retinoic acid resistance by suppressing cellular retinoic acid-binding protein (CRABP2) transcription in HER2-enriched breast cancers [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 1401.
Abstract 1446: GRP78 targeting peptides deliver liposomal doxorubicin specifically to cancer and enhance the efficacy of radiation therapy
Authors: Singh Abhay K. , Shyam Chandresh , Lewis Calvin , Zhang Wendy , Deore Sapna , Kapoor Vaishali , Hallahan Dennis E.
Published: July 2021

Traditional radiation therapy is often associated with significant toxicity to normal cells that could limit cancer therapy's success. The greater understanding of the molecular differences between cancer cells and normal cells has led to targeted therapies in cancer treatment. We discovered the radiation-inducible expression of Glucose regulated protein 78kDa (GRP78) in cancer using phage-display peptide libraries. In this study, we screened a peptide library and discovered novel peptides targeting GRP78. The top ten peptides were further evaluated for their affinity of binding to recombinant GRP78 using surface plasmon resonance technology. Since GRP78 is induced on cancer cells' surface, we irradiated the lung cancer cell lines A549 and H460 to evaluate peptide binding. Flow cytometry analysis of Dylight 488 conjugated peptides led to identifying peptides A and K with the highest binding level to irradiated A549 and H460. Peptides A and K were compared for their efficacy to deliver liposomal doxorubicin specifically to tumors. Copper-free click-chemistry was used to conjugate peptides to the liposome surface. Whole-body near-infrared imaging was performed to evaluate the biodistribution of the peptide conjugated liposomes in nude mice bearing A549 and H460 tumors. Both peptides A and K specifically delivered liposomes to the tumors compared to the non-targeted liposomes. In pilot tumor growth experiments, we found that GRP78 targeting peptide conjugated liposomes delayed the growth of tumors in nude mice compared to non-targeted liposomes. The inhibition of tumor growth was further enhanced in combination with radiation, indicating efficient delivery of the radiation-sensitizer doxorubicin specifically to tumors. Currently, studies are underway to evaluate the microscopic biodistribution of doxorubicin in tumor sections. Mass spectrometry analysis of various organs will be performed to determine the pharmacokinetics of liposomal doxorubicin delivered by GRP78 targeting peptides. In conclusion, our novel GRP78 targeting peptides specifically deliver liposomal doxorubicin to tumors. Doxorubicin encapsulated in GRP78 targeting peptide conjugated liposomes enhances the tumor growth inhibitory activity of radiation therapy. Specific delivery of radiation-sensitizing drugs encapsulated in a liposome by peptides that target radiation-inducible GRP78 is a promising therapeutic strategy for various cancers.

Citation Format: Abhay K. Singh, Chandresh Shyam, Calvin Lewis, Wendy Zhang, Sapna Deore, Vaishali Kapoor, Dennis E. Hallahan. GRP78 targeting peptides deliver liposomal doxorubicin specifically to cancer and enhance the efficacy of radiation therapy [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 1446.
Abstract 1470: Preclinical activity of the first reversible, potent and selective inhibitor of EGFR exon 20 insertions
Authors: Siegel Franziska , Siegel Stephan , Graham Keith , Kaplan Bethany , Petersen Kirstin , Boemer Ulf , Eberspaecher Uwe , Korr Daniel , Moenning Ursula , Suelzle Detlev , Schroeder Jens , Prinz Florian , Zitzmann-Kolbe Sabine , Karsli-Uzunbas Gizem , Lewis Timothy , Hermsen Mario , Cherniack Andrew , von Nussbaum Franz , Eis Knut , Meyerson Matthew , Greulich Heidi
Published: July 2021

Several targeted therapeutic options exist for non-small cell lung cancer (NSCLC) patients whose tumors harbor mutations of EGFR, including L858R, exon 19 deletions, and the acquired resistance mutation, T790M. However, there is no approved targeted therapy for patients with EGFR exon 20 insertions, highlighting an ongoing unmet medical need. Here, we describe a potent and selective inhibitor of EGFR exon 20 insertion mutants with decreased activity on the wild-type EGFR for an improved therapeutic window. Our compound, BAY-568, exhibits greater than 20-fold selectivity for EGFR exon 20 insertions compared to wild-type EGFR in isogenic Ba/F3 models and in cancer cell lines endogenously expressing EGFR exon 20 insertions. This activity is also observed in xenograft models in vivo, correlating with reductions in phospho-EGFR and phospho-Erk in tumors but not skin samples from treated mice. BAY-568 furthermore has even greater activity towards the “classical” erlotinib-sensitive mutations, L858R and exon 19 deletions. Importantly, our compound is reversible, differentiating it from other investigational compounds currently in clinical trials. Consistent with this, the presence of a C797S mutation, typically found in patients with acquired resistance to osimertinib, has no effect on the activity of BAY-568. Taken together, these results demonstrate the ability of BAY-568 to kill cancer cells harboring exon 20 insertions and other EGFR mutations with decreased activity on wild-type EGFR, irrespective of C797S mutation status.

Citation Format: Franziska Siegel, Stephan Siegel, Keith Graham, Bethany Kaplan, Kirstin Petersen, Ulf Boemer, Uwe Eberspaecher, Daniel Korr, Ursula Moenning, Detlev Suelzle, Jens Schroeder, Florian Prinz, Sabine Zitzmann-Kolbe, Gizem Karsli-Uzunbas, Timothy Lewis, Mario Hermsen, Andrew Cherniack, Franz von Nussbaum, Knut Eis, Matthew Meyerson, Heidi Greulich. Preclinical activity of the first reversible, potent and selective inhibitor of EGFR exon 20 insertions [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 1470.
Abstract 1499: Targeting Ras mutation with TCR therapy
Authors: Mou Nan , Yu Yue , Yuan Jijun
Published: July 2021

Ras oncogenes (K/N/HRAS) are frequently mutated in 20% of all human cancers. Globally, approximately 3.4 million new cancer cases are diagnosed annually carrying Ras mutations. There is a large unmet medical need for treating cancers carrying Ras mutations. KRAS mutations account for around 80% of total Ras mutations. Codon 12 and 13 are most frequently mutated in KRAS oncogene. RasG12V and RasG13D represent approximately 22-58% cases of KRAS mutations. RAS mutations are immunogenic and can be recognized by T cell receptors in HLA restricted manner (Steven A. Rosenberg, 2016). Here we report identification of two human TCRs specifically recognizing RasG12V and RasG13D mutations isolated from tumor infiltrating lymphocytes of colorectal cancer patients. Both TCRs show high avidity to RasG12V and RasG13D mutations presented by MHCII alleles widely distributed in human population. Engineered B13.14.1 TCR-T and B8.2.4 TCR-T recognize RASG12V and RASG13D presented on HLA matched tumor cell line and induce strong INFγ release. Furthermore, potent yet specific tumor cell lysis was observed in multiple RASG12V and RASG13D tumor cell lines. Our RasG12V and RasG13D targeted TCR-T therapy could hold great potential for treatment of oncogenic Ras mutation driven cancers.

Citation Format: Nan Mou, Yue Yu, Jijun Yuan. Targeting Ras mutation with TCR therapy [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 1499.
Abstract 1535: Allogenic CAR iNKT as a cell therapy platform targeting tumor antigen Isomesothelin
Authors: Gopisetty Anupama , Chen Yibin , Nguyen Quynh , Chiriva-Internati Maurizio
Published: July 2021

Effective chimeric antigen receptor (CAR) T-cell therapy targeting B-cell malignancies has paved the way for alternate strategies targeting cancer. Current active research is directed towards development of safe, allogeneic off the shelf cell (OTS) therapy products. This could be potentially a step forward in the targeted cancer immunotherapy field. Invariant NKT cells (iNKT) are deemed as one of the unconventional T-cell populations with semi invariantly re-arranged TCR. They recognize lipid antigens Via CD1d, an MHC Class 1 like molecule. Recognition of CD1d expressed on various hematopoietic cells is important for targeted tumor specific iNKT cytotoxicity in various leukemia, lymphoma malignancies. Through the recent years, CAR-iNKT adoptive cell therapy has drawn excitement and interest because of their intrinsic anti-tumor property. More specifically CAR-iNKTs have been shown to target solid tumors such as neuroblastoma in preclinical studies and proceeded to phase 1 clinical trials. The innate ability of iNKT cells to kill tumor cells and also cause very less GVHD provides an extremely attractive platform for developing OTS product. Allogeneic CAR iNKT cells have been less explored for cancer immunotherapy. Although the relative percentage of iNKTs are very low in peripheral blood (~0.01% of T-lymphocytes), we report an efficient method of enrichment and expanding a large and pure population of iNKTs and genetically modify them for targeting solid tumor antigens such as Isomesothelin (IsoMSLN), an alternative splice variant of mesothelin. Many pre-clinical studies have been assessed targeting mesothelin, a tumor associated antigen overexpressed in malignant cells. A safe allogenic CAR targeted therapy for IsoMSLN has yet to be demonstrated. iNKTs expanded and transduced with our novel protocol targeting IsoMSLN, are highly cytotoxic and suggests a central memory phenotype that could potentially persist longer in vivo.

Citation Format: Anupama Gopisetty, Yibin Chen, Quynh Nguyen, Maurizio Chiriva-Internati. Allogenic CAR iNKT as a cell therapy platform targeting tumor antigen Isomesothelin [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 1535.
Abstract 1560: Targeting RNA-binding protein HuR to improve anti-PD-1 immunotherapy response in breast cancer
Authors: Zhang Qi , Wu Xiaoqing , Lan Lan , Wei Lanjing , Xu Liang
Published: July 2021

Breast cancer has been considered as a less immune-sensitive disease. Patients with metastatic breast cancer have a particularly low objective response rate (ORR) to anti-PD-1/PD-L1 therapy (3%~24%). It will be valuable to improve the treatment efficacy of anti-PD-1 immunotherapy in breast cancer. Recent studies reported that exosomal PD-L1 derived from tumor cells mediated immune surveillance evasion by interacting with the PD-1 receptor on T cells and contributed to the low ORR in anti-PD-1/PD-L1 immunotherapy.The RNA-binding protein Hu antigen R (HuR) is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs, including PD-L1 mRNA. HuR also plays an important role in the exosome secretion process by regulating RAB27B mRNA. In this study, we aim to identify HuR biological functions in the cancer-immune system and evaluate the combination strategy using a HuR inhibitor with an anti-PD-1 antibody in breast cancer. We show here the direct binding of HuR protein to PD-L1 mRNA in human breast cancer cell line MDA-MB-231 by Ribonucleoprotein immunoprecipitation (RNP-IP). We also show that PD-L1 protein expression level is decreased in HuR knockout clones and in exosomes derived from HuR knockout clones. HuR knockout downregulates exosome secretion with decreased size in MDA-MB-231 cells. KH-3 is a small molecule identified from several chemical libraries that disrupts the binding of HuR and its target mRNAs.To test the combination strategy using a HuR inhibitor and anti-PD-1 antibody in vivo, we established the EMT6 orthotopic mouse breast cancer model. A combination treatment with HuR inhibitor KH-3 and anti-PD-1 antibody inhibits tumor growth and prolongs survival in this breast cancer model. In conclusion, we preliminarily explored the role of HuR in the cancer-immune system, we showed that HuR may be a promising therapeutic target to overcome immune evasion and improve immunotherapy response in breast cancer.

Citation Format: Qi Zhang, Xiaoqing Wu, Lan Lan, Lanjing Wei, Liang Xu. Targeting RNA-binding protein HuR to improve anti-PD-1 immunotherapy response in breast 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 1560.
Abstract 1573: Combinatorial antitumor effects of CD3-based trispecific T cell activating constructs (TriTACs) and checkpoint inhibitors in preclinical models
Authors: Molloy Mary Ellen , Valenzuela Laura B. , Basak Priyanka E. , Law Che-Leung
Published: July 2021

Tri-specific T Cell-Activating Constructs (TriTACs) are T-cell engagers that have been developed to redirect T cells to kill tumor cells. TriTAC molecules are currently being investigated in multiple phase 1/2 clinical trials in solid tumors, including HPN424 targeting prostate-specific membrane antigen (PSMA) in prostate cancer (NCT03577028), HPN536 targeting mesothelin (MSLN) in multiple malignancies (NCT03872206) and HPN328 targeting Delta Like Canonical Notch Ligand 3 (DLL3) in small cell lung cancer (SCLC) (NCT04471727). TriTAC molecules consist of a single domain antibody (sdAb) specific for a tumor antigen, a sdAb specific for serum albumin for half-life extension, and a single chain Fv (scFv) specific for the CD3ϵ subunit of the T cell receptor (TCR) complex for T cell engagement. In the presence of antigen expressing tumor cells, TriTAC treatment leads to potent T cell activation, production of proinflammatory cytokines, and re-directed tumor cell killing. Upregulation of co-stimulatory and/or inhibitory receptors during activation can potentially affect the cytolytic functions of TriTAC-activated T cells. We demonstrate that PD-1 can be readily detected on T cells subsequent to the engagement of the TCR by the TriTAC molecules HPN424, HPN536, and HPN328 in the presence of tumor cells expressing the target antigens PSMA, MSLN, and DLL3, respectively. We also demonstrate that TriTAC molecules upregulate PD-L1 on T cells in a dose-dependent manner. While induction of PD-1 on T cells suggests that cytolytic activity of TriTAC-activated T cells may be down-modulated by PD-L1-expressed by target tumor cells, simultaneous upregulation of PD-L1 on T cells suggests a second mechanism through which the functions of TriTAC-activated T cells could be further regulated by the PD1/PD-L1 axis in the tumor microenvironment through T cell-T cell interactions. In addition, in the MSLN-expressing NCI-H292 lung cancer model that co-expresses constitutive, high levels of PD-L1, both anti-PD1 and anti-PDL1 antibodies significantly enhanced the antitumor effects of the MSLN-targeting TriTAC HPN536 in vivo. Together these results demonstrate the potential utility of PD1/PDL1 blockade to enhance the potency of TriTAC-mediated tumor cell killing, supporting further investigation of these combinatorial approaches in patients.

Citation Format: Mary Ellen Molloy, Laura B. Valenzuela, Priyanka E. Basak, Che-Leung Law. Combinatorial antitumor effects of CD3-based trispecific T cell activating constructs (TriTACs) and checkpoint inhibitors in preclinical 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 1573.
Abstract 1595: Small molecule inhibition of the ubiquitin ligase CBL-B results in potent T and NK cell mediated anti-tumor response
Authors: Rountree Ryan , Cohen Frederick , Tenn-McClellan Austin , Borodovsky Alexandra , Gallotta Marilena , Stokes Jennifer , Romo Jose Gomez , Karim Chris , Hansen Gwenn M. , Guiducci Cristiana , Sands Arthur , Gosling Jennifa
Published: July 2021

The E3 ubiquitin ligase Casitas B-lineage lymphoma b (CBL-B) is expressed in T cells where it functions as an important negative regulator of immune activation. CBL-B d attenuates T-cell activation initiated by TCR engagement in part by mediating the requirement for CD28 co-stimulation, thus setting the threshold for T cell activation. CD4+ and CD8+ T cells from mice deficient in cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN-γ when stimulated ex vivo with anti-CD3. Cbl-b deficient mice also demonstrate enhanced NK cell function. Here we describe NX-1607, an investigational, orally bioavailable, small molecule inhibitor of CBL-B. NX-1607 demonstrates potent biochemical inhibition of CBL-B leading to stimulatory effects on human immune cells at low nanomolar concentrations. NX-1607 induction of IL-2 and IFN-γ secretion occurs in primary human T cells stimulated with anti-CD3 antibodies, in both the presence and absence of CD28 co-stimulation, although to a lesser degree in the absence of co-stimulation. In vivo, oral administration of NX-1607 in mice demonstrated significant tumor growth inhibition in two colon carcinoma tumor models, CT26 and MC38, as well as a triple negative breast tumor model, 4T1. The change in tumor microenvironment caused by NX-1607 treatment leads to rapid NK cell infiltration followed by infiltration of activated CD8+ T cells. Depletion of CD8+ T cells or NK cells completely abrogated NX-1607 antitumor activity. Importantly, the combination of NX-1607 and anti-PD-1 can substantially increase the median overall survival and the frequency of complete tumor rejections in all three tumor models. These studies provide significant insights into the antitumor activity of this novel, small molecule E3 ligase inhibitor and deliver experimental support for clinical development of NX-1607 given as monotherapy or in combination with PD-1 blockade.

Citation Format: Ryan Rountree, Frederick Cohen, Austin Tenn-McClellan, Alexandra Borodovsky, Marilena Gallotta, Jennifer Stokes, Jose Gomez Romo, Chris Karim, Gwenn M. Hansen, Cristiana Guiducci, Arthur Sands, Jennifa Gosling. Small molecule inhibition of the ubiquitin ligase CBL-B results in potent T and NK cell mediated anti-tumor response [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 1595.
Abstract 1712: Neospora caninum: An immunotherapeutic protozoan against cancer
Authors: Lantier Louis , Poupée-Beaugé Agathe , Tommaso Anne Di , Ducournau Céline , Germon Stéphanie , Lee Gordon S. , Touze Antoine , moiré Nathalie , Dimier-Poisson Isabelle
Published: July 2021

Background: Immunotherapy induces, provides, and/or reactivates anti-tumor immune responses. Some microorganisms also can initiate response that lyzes infected tumor and/or stimulates systemic immunity. Attenuated viruses or bacteria are well studied as oncotherapeutics, but no protozoa except Toxoplasma gondii. We assessed the effect on tumors of other protozoa that were naturally non-pathogenic to humans. Thus, we discovered the ability to use Neospora caninum (Nc) in a manner and form that demonstrated a synergistic array of pertinent immunotherapeutic characteristics against solid cancers. We report on the most recent data notably from Nc engineered to secrete human IL-15 within the tumor.

Methods: In vitro, the immunostimulatory properties of Nc strains wildtype and engineered to secrete human IL-15 were studied. In vivo experiments of treatment with of Nc tachyzoites administered locally (intra and peri tumoral) or remotely (subcutaneous) in a murine thymoma EG7 tumor and in human Merkel cell carcinoma (MCC).

Results: We demonstrated that the treatment of thymoma EG7 by Nc strongly inhibited tumor development. Analysis of immune responses and interactions between Nc and tumor cells showed that Nc had the ability to lyze infected cancer cells, reactivated immune competence within the tumor microenvironment (TME), and activated the systemic immune system by promoting the recruitment of immune cells to the site of tumor. We also established in a NOD/SCID mouse model that Nc was able to induce a strong regression of human MCC. Recently, to further enhance oncotherapeutic effect, we engineered an Nc strain to secrete human IL-15 (cross reactive with mouse cells), associated with alpha subunit of IL-15 receptor, increasing its stability. This strain induced proliferation of human PBMCs and their secretion of IFN-γ. In the EG7 model, human IL-15 secreting Nc showed greater protection against tumor development, confirming enhancement of immunotherapy by engineering Nc to deliver/secrete IL-15.

Conclusion: These results highlight Neospora caninum as a potentially extremely efficient, and non-toxic anti-cancer agent, capable of being engineered to express at its surface or to secrete bio-drugs, like human IL-15 cytokine. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine capable of vectoring molecular therapy, overcoming TME defenses.

Citation Format: Louis Lantier, Agathe Poupée-Beaugé, Anne Di Tommaso, Céline Ducournau, Stéphanie Germon, Gordon S. Lee, Antoine Touze, Nathalie moiré, Isabelle Dimier-Poisson. Neospora caninum: An immunotherapeutic protozoan against 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 1712.