bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2602: Combined inhibition of AXL and ATR enhances cell death and immune response in small cell lung cancer
Authors: Ramkumar Kavya , Tanimoto Azusa , Stewart C. Allison , Wang Qi , Shen Li , Cardnell Robert J. , Rodriguez B. Leticia , Gibbons Don L. , Wang Jing , Gay Carl M. , Byers Lauren A.
Published: June 2022

Small cell lung cancer (SCLC) is an aggressive lung tumor of neuroendocrine origin with a dismal 5-year survival rate. Despite high response rates to initial therapy, rapid development of therapeutic resistance limits overall survival. There are also limited treatment options, particularly in the relapsed setting. Our group and others have shown that AXL, a TAM family receptor tyrosine kinase that is highly expressed in mesenchymal tumors, mediates resistance to chemotherapy, radiation and targeted therapies in SCLC, non-small cell lung cancer and other cancers, through its roles in driving epithelial to mesenchymal transition and DNA damage repair. AXL has also been implicated in immune escape. Based on these findings, we hypothesize that AXL targeting may be a potential therapeutic approach in SCLC. We screened BGB324 (bemcentinib), a selective small-molecule AXL inhibitor in clinical trials for various advanced solid tumors, in a panel of 60 human SCLC cell lines using cell viability assays. The SCLC cell lines showed a range of sensitivities, with IC50 values ranging from 41 nM to 10 µM (median IC50 3.1 µM) with NeuroD1-driven SCLC cell lines being highly sensitive to BGB324 (ANOVA p<0.05). BGB324 also showed potent inhibition of tumor growth in an in vivo SCLC model. BGB324 further combined synergistically with an ATR inhibitor (AZD3738) and induces significant DNA damage. In a syngeneic model of SCLC, the combination of BGB324, AZD6738 and an anti-PDL1 antibody combination also induced significant tumor regression. Together, these promising findings show that AXL inhibition may be effective in SCLC and support further investigation of AXL and ATR inhibitor combinations with immune checkpoint blockade.

Citation Format: Kavya Ramkumar, Azusa Tanimoto, C. Allison Stewart, Qi Wang, Li Shen, Robert J. Cardnell, B. Leticia Rodriguez, Don L. Gibbons, Jing Wang, Carl M. Gay, Lauren A. Byers. Combined inhibition of AXL and ATR enhances cell death and immune response in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2602.
Abstract 2603: USP1 inhibitor synthetic lethality in BRCA1-mutant cancer is driven by PCNA ubiquitination
Authors: Engel Justin , Bandi Madhavi , Simoneau Antione , Lazarides Katherine , Gotur Deepali , Pham Truc , Liu Shangtao , Meier Samuel , Choi Ashley , Zhang Hongxiang , Shen Binzhang , Li Fang , Whittington Douglas , Gong Shanzhong , Pan Xuewen , Yu Yi , Gu Lina , Throner Scott , Maxwell John , Chen Yingnan , Huang Alan , Andersen Jannik , Feng Tianshu
Published: June 2022

CRISPR-based functional genomic screening is a powerful approach for identifying novel classes of synthetic lethal drug targets. Here, we define the deubiquitinase USP1 as a synthetic lethal target in cancers with underlying DNA repair vulnerabilities. A highly potent and selective small molecule USP1 inhibitor conferred a viability defect in BRCA1-mutant, but not WT cell lines by activating replication stress. Genome-wide CRISPR screening uncovered RAD18 and UBE2K, which promote PCNA mono- and poly-ubiquitination respectively, as key mediators of USP1-BRCA1 dependency. Increased cellular mono- and poly-ubiquitination reduced PCNA protein levels, and restoration of PCNA protein expression rescued USP1 inhibitor sensitivity. USP1 dependency is associated with upregulated RAD18 and UBE2K expression, suggesting that elevated PCNA ubiquitination in the context of BRCA1 deficiency mediates USP1 synthetic lethality. Interestingly, USP1, but not PARP1 inhibition, elicited a viability defect in a subset of BRCA1/2 WT lung cancer cell lines, indicative of novel synthetic lethal interactions unique to USP1. Moreover, dual inhibition of PARP1 and USP1 are strongly synergistic in PARP1 inhibitor-responsive cell line models. Strong in vivo anti-tumor activity across multiple tumor models was demonstrated with USP1 inhibition alone and in combination with the PARP1 inhibitor olaparib. Our studies suggest that USP1 and PARP1 inhibitors target BRCA1-mutant cancer though distinct yet synergistic mechanisms. As such, USP1 inhibitors may provide novel treatment strategies for PARP1 inhibitor-resistant and -naïve BRCA1-mutant cancer.

Citation Format: Justin Engel, Madhavi Bandi, Antione Simoneau, Katherine Lazarides, Deepali Gotur, Truc Pham, Shangtao Liu, Samuel Meier, Ashley Choi, Hongxiang Zhang, Binzhang Shen, Fang Li, Douglas Whittington, Shanzhong Gong, Xuewen Pan, Yi Yu, Lina Gu, Scott Throner, John Maxwell, Yingnan Chen, Alan Huang, Jannik Andersen, Tianshu Feng. USP1 inhibitor synthetic lethality in BRCA1-mutant cancer is driven by PCNA ubiquitination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2603.
Abstract 261: Released exosomes from colon cancer cells regulate formation and progression of perineural invasion
Authors: Knoblich Colewyn , Rezvani Khosrow
Published: June 2022

Colorectal cancer (CRC) is the third most diagnosed cancer and cause of cancer related mortality. CRC that metastasizes to distance sites results in poor prognosis, diminished survival, and reduced effectiveness of treatments. Perineural invasion (PNI) is an emerging field that aims to understand cancer-neuronal crosstalk that enables cancer to spread via nerves. Several studies have indicated that tumor derived exosomes (TDEs) play significant roles in the initiation and spread of cancer, along with driving chemoresistance. We hypothesize that CRC derived exosomes alter nerves within the surrounding environment and result in recruitment of nerves towards tumors for invasion along the nerve. Exosomes from both HCT116 and SW837 colon cancer cells were isolated and purified using a modified iodixanol ultracentrifugation gradient technique. A Proteomic analysis conducted on cancer derived exosomes revealed several potential proteins involved in neuronal growth and migration, including Vinculin (VCL), Periostin (POSTN or OSF-2), DCLK1, and USP9X. In another set of experiments, conditioned media (CM) from HCT116 and SW837 cells were added to both PC12 neuronal-like cells and dorsal root ganglion (DRG) from C57BL/6J mice to understand the effects of excreted cancer factors on neuronal outgrowth and differentiation. CM studies showed how signaling factors excreted from colon cancer cells induce increased nerite outgrowth in both PC12 cells and DRG. These results were not seen in CM from cancer cells that were grown in the presence of an exosome inhibitor. Altogether, these results indicate that CRC utilizes exosomes to alter the adjacent neuronal networks as a means to employ surrounding nerves for invasion.

Citation Format: Colewyn Knoblich, Khosrow Rezvani. Released exosomes from colon cancer cells regulate formation and progression of perineural invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 261.
Abstract 2623: Variants of CLIP-170 associated with taxane resistance in tumors
Authors: Thakkar Prashant V. , Kita Katsuhiro , del Castillo Urko , Stone William G. , Galletti Giuseppe , Madhukar Neel , Navarro Elena Vila , Barasoain Elena , Goodson Holly V. , Sackett Dan , Díaz José Fernando , Lu Yao , RoyChoudhury Arindam , Molina Henrik , Elemento Olivier , Shah Manish A. , Giannakakou Paraskevi
Published: June 2022

Despite its widespread use, the majority of patients with gastric cancer (GC) will not respond to taxane chemotherapy due to resistance mechanisms. Recently, we reported the discovery of a novel truncated variant of the microtubule plus-end binding protein CLIP-170, hereafter CLIP-170S, whose expression is enriched in taxane resistant cell lines and patients with GC. Mass-spec proteomics and 5’-RACE further showed that CLIP-170S lacked the first 155 amino acids, including the Cap-Gly motif required for microtubule plus-end localization. CLIP-170S knockdown reversed taxane resistance in cells and xenografts, whereas its re-expression led to resistance, suggesting causation. Unlike canonical CLIP-170, we showed that CLIP-170S was mislocalized from the MT plus-end to the MT lattice. Computational analysis of RNA-seq data in conjunction with the connectivity map from taxane-sensitive and resistant GC cell lines, predicted imatinib as the top candidate drug to overcome drug resistance. Imatinib treatment completely reversed taxane resistance, as predicted, and did so unexpectedly by selective depletion of CLIP-170S. Other RTK inhibitors also depleted CLIP-170S, suggesting a class effect. We are currently unraveling the molecular mechanisms by which a) CLIP-170S impairs taxane association to its microtubule binding site and b) Imatinib and other RTK inhibitors selectively deplete CLIP-170S. Our data show that CLIP-170S is a clinically prevalent variant that confers taxane resistance in tumors, whereas the discovery of Imatinib as a CLIP-170S inhibitor provides novel therapeutic opportunities for future trials.

Citation Format: Prashant V. Thakkar, Katsuhiro Kita, Urko del Castillo, William G. Stone, Giuseppe Galletti, Neel Madhukar, Elena Vila Navarro, Elena Barasoain, Holly V. Goodson, Dan Sackett, José Fernando Díaz, Yao Lu, Arindam RoyChoudhury, Henrik Molina, Olivier Elemento, Manish A. Shah, Paraskevi Giannakakou. Variants of CLIP-170 associated with taxane resistance in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2623.
Abstract 263: PIM kinase regulates actin dynamics in hypoxia
Authors: Jensen Corbin C. , Warfel Noel A.
Published: June 2022

One of the leading factors in prostate cancer mortality is the onset of metastatic events. This is seen by the drastic decline in survival rates following tumor escape from the primary site. A key promoter of metastasis is tumor hypoxia, or low oxygen concentration. Our lab previously demonstrated that Proviral Integration site for Moloney murine leukemia virus (PIM)-1, a serine-threonine kinase, is upregulated in hypoxia and that inhibition of PIM1 reduced metastatic occurrence in orthotopic mouse models. Here, we identify a novel signaling pathway that links hypoxia and PIM kinase to the actin cytoskeleton and cell motility. An unbiased screen to discover new PIM substrates in hypoxia identified Abl-Interactor 2 (Abi2), an integral member of the actin regulating Wave Regulatory Complex (WRC), as a novel PIM kinase substrate. Biochemical analysis demonstrates that phosphorylation of Abi2 at our identified PIM sites increases Abi2 stability. Overexpression of PIM1 increases Abi2 and other WRC members at the protein level, resulting in increased actin protrusions and cell motility. CRISPR-generated Abi2 knockout cell lines showed diminished protrusive activity and negated both the necessity and sufficiency of PIM kinase to increases protrusive activity in cancer cells. Lastly, in vivo experiments show that overexpression of PIM1 increases tumor invasion into the surrounding tissue in an Abi2-dependent manner, and small molecule PIM inhibitors significantly reduce invasion. In summary, we uncovered a novel role for PIM1 in promoting cellular motility and protrusion, identified novel phosphorylation sites that control the stability of Abi2 in normoxic and hypoxic conditions, and highlight a novel PIM1/Abi2 signaling axis that regulates hypoxia-induced tumor invasion. This research provides important insight in the role of PIM signaling in prostate tumors and yields preclinical evidence for further use of PIM kinase inhibitors as a means to inhibit tumor invasion, a key driving step in the formation of lethal prostate cancer metastasis.

Citation Format: Corbin C. Jensen, Noel A. Warfel. PIM kinase regulates actin dynamics in hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 263.
Abstract 2635: Inhibiting deoxycytidine kinase significantly inhibits tumor growth in xenograft models of castration resistant prostate cancer
Authors: Radu Caius G. , Schultz Kenneth A. , Lückerath Katharina , Shepard H Michael , Czernin Johannes
Published: June 2022

Mammalian cells synthesis deoxyribonucleoside triphosphate (dNTP) precursors for DNA replication via the de novo pathway (DNP), and to a lesser extent, via the nucleoside salvage pathway (NSP). Evidence suggests that rapid tumor cell growth associated with increased dNTP needs results in elevated NSP activity. As deoxycytidine kinase (dCK) is the rate-limiting enzyme of the NSP, and the only enzyme that can supply cells with all dNTP precursors for DNA replication, dCK has been implicated as a promising therapeutic target. Data from the Cancer Cell Line Encyclopedia demonstrated that most human cancer cell lines have elevated dCK gene expression (2- to 17-fold) therefore suggesting selective susceptibility of tumor cells to dCK inhibition. Accordingly, a potent first-in-class oral dCK inhibitor, TRE-515, was evaluated as a single agent in two human castration-resistant prostate cancer (CRPC) models. In the first CRPC model, C4-2, TRE-515 was administered intraperitoneally once a day for eight consecutive days (50 mg/kg, QDx8). TRE-515 significantly inhibited tumor growth (50.7%, p=0.021, relative to vehicle control), with 50% of the TRE-15 treated tumors regressing during the eight-day treatment window. In a repeat C4-2 study, TRE-515 monotherapy trended toward tumor growth inhibition, (53.7%, p=0.061, relative to vehicle control). A second CRPC model, 22Rv1, was then selected as it has been shown to carry DNP mutations, suggesting enhanced sensitivity to dCK inhibition. In this study, TRE-515 was dosed orally two times a day for 28 days (75 mg/kg, BIDx28) and plasma deoxycytidine (dC) concentrations (a substrate for dCK) were measured 7 hours after the morning dose on day 21. TRE-515 alone significantly inhibited tumor growth (49.9%, p=0.0004, relative to vehicle control), and growth inhibition was associated with a 77% increase in plasma dC levels (p<0.0001), relative to vehicle control, consistent with TRE-515 inhibitory activities on dCK. These data are in agreement with a previously reported study on target enzyme inhibition by TRE-515 which employed a clinically validated PET probe imaging of dCK. Taking together these results support the therapeutic potential for TRE-515 to selectively inhibit cancer cells that rely on dCK for DNA synthesis and rapid malignant proliferation. As such, TRE-515 is currently being evaluated in a phase 1 open-label, dose escalation study in solid tumors (NCT #05055609).

Citation Format: Caius G. Radu, Kenneth A. Schultz, Katharina Lückerath, H Michael Shepard, Johannes Czernin. Inhibiting deoxycytidine kinase significantly inhibits tumor growth in xenograft models of castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2635.
Abstract 2637: Inhibition of novel kinase MAP3K19 reverses EMT and kills triple negative breast cancer cell lines
Authors: Nguyen Khoa , Cheng Thomas , Alzoubi Madlin , Elliott Steven , Lee Sean , Drewry David , Collins-Burow Bridgette , Brognard John , Burow Matthew
Published: June 2022

The Triple-Negative Breast Cancer (TNBC) subtype is a breast cancer subpopulation whose phenotype lacks estrogen and progesterone growth receptors, and lacks HER2 amplification. This contrasts with the Luminal and HER2+ subpopulations for which clinical precision therapies exist to target cancer proliferation and improve prognoses. Consequently, the discovery of new targets is necessitated by the TNBC subtype, which has the poorest patient outcomes. Our studies have identified MAP3K19 as one such potential target. MAP3K19 an understudied protein kinase with poorly characterized physiological functions outside of its roles in the Mitogen-Activated Protein Kinase (MAPK) signaling cascade. Broadly, the MAPK signaling has been associated with cell proliferation and differentiation in pathways both intracellular and extracellular, which in the context of cancer cell biology warrants investigation into cancer cell behavior as well as the associated tumor microenvironment (TME). The Published Kinase Inhibitor Set (PKIS) is an open-access drug panel designed by GlaxoSmithKline to identify novel kinases. Preliminary PKIS cell-viability screening identified GSK1440913, GW494601, and GSK907232A as compounds with the most adverse effects on cell phenotype and growth in TNBC lines MDA-MB-231 and PDX-derived TU-BcX-49S. Analysis of common targets revealed MAP3K19 a common target, suggesting its importance in cancer cell growth and proliferation. AT-9283, a more specific MAP3K19 inhibitor, was used to support the initial results. Treatment with AT-9283 resulted in a dose-dependent killing effect. Furthermore, MAP3K19 inhibition resulted in decreased EMT gene signatures by qPCR. To summarize, MAP3K19 was identified by the PKIS screen as a major kinase in TNBC. Treatment with AT-9283, a more specific MAP3K19 inhibitor, resulted in cellular death and reversal in EMT gene signature by qPCR. Future directions for this project involve genetic knock-down and overexpression of MAP3K19 to evaluate its roles in biology and its signaling mechanisms.

Citation Format: Khoa Nguyen, Thomas Cheng, Madlin Alzoubi, Steven Elliott, Sean Lee, David Drewry, Bridgette Collins-Burow, John Brognard, Matthew Burow. Inhibition of novel kinase MAP3K19 reverses EMT and kills triple negative breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2637.
Abstract 2677: JZP815, a potent and selective pan-RAF inhibitor, demonstrates efficacy in RAF and RAS mutant tumor pre-clinical models
Authors: Hauptschein Robert , Woodcock Simon , Belfield Andrew , Mason Helen , Keppel Dorottya , Markova Svetlana , Kim Kyuri , Roller Shane , Phillips Caroline , Jones Clifford D. , Humphreys Robin C.
Published: June 2022

Activation of the mitogen-activated protein kinase (MAPK) pathway by oncogenic mutations in RAS and RAF is a frequent driver of human cancer. Targeting specific components of the pathway is a precise and rational route to deliver benefits to cancer patients with high unmet therapeutic needs. The RAF kinase proteins (ARAF, BRAF, CRAF) are core pathway components which mediate MAPK signaling. Mutations in these critical signaling proteins leads to constitutive activation of the MAPK pathway and tumor growth. Using state of the art screening methodologies and medicinal chemistry we discovered and developed a next generation pan-RAF kinase inhibitor, JZP815, which potently inhibits wild-type and mutant RAF kinases in cell-free and cell-based assays. JZP815 is a selective RAF kinase inhibitor that suppresses all 3 RAF kinase family members at low-to-sub nanomolar potencies in cell-free assays. Moreover, in tumor cells JZP815 does not induce significant paradoxical pathway activation, observed with approved 1st generation BRAF-selective inhibitors, while demonstrating equivalent potencies for MAPK pathway inhibition driven by either mutant RAF monomers or RAS-induced RAF dimers in tumor cells. JZP815 significantly inhibited tumor growth as a single agent in multiple mouse xenograft solid tumor models harboring RAS and/or BRAF mutations. Furthermore, JZP815’s favorable pharmacokinetic profile sustained on-target pathway pharmacodynamic responses in a predictable dose and time dependent manner. JZP815 demonstrated enhanced activity when combined with inhibitors of other MAPK pathway components in both class 2 and class 3 mutant BRAF patient-derived tumor cells ex vivo, and KRAS mutant NSCLC and CRC xenografts in vivo. In summary, JZP815 is a potent and selective pan-RAF inhibitor that is orally bioavailable in multiple preclinical species with a well-behaved safety profile and pharmacokinetic properties predicted to provide drug exposure required for target engagement in humans, and therefore suitable for advancing into first-in-human trials.

Citation Format: Robert Hauptschein, Simon Woodcock, Andrew Belfield, Helen Mason, Dorottya Keppel, Svetlana Markova, Kyuri Kim, Shane Roller, Caroline Phillips, Clifford D. Jones, Robin C. Humphreys. JZP815, a potent and selective pan-RAF inhibitor, demonstrates efficacy in RAF and RAS mutant tumor pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2677.
Abstract 2678: LLL12B, a novel small molecule STAT3 inhibitor induces apoptosis and suppresses cell migration and tumor growth in triple-negative breast cancer cells
Authors: Pan Li , Chen Xiang , Rassool Feyruz V. , Li Chenglong , Lin Jiayuh
Published: June 2022

Persistent STAT3 signaling has been recognized as a cancer therapeutic target in some cancer types including triple-negative breast cancer, and the development of effective small molecule STAT3 inhibitors for potential cancer therapy is desirable. In this study, we tested a novel oral bioavailable small molecule STAT3 inhibitor, LLL12B in MDA-MB-231, SUM159, and 4T1 triple-negative breast cancer cells. Triple-negative breast cancer cells frequently express persistent STAT3 phosphorylation and its cell viability is sensitive to STAT3 knockdown by siRNA. LLL12B selectively inhibited IL-6 mediated induction of STAT3 phosphorylation but had little effect on IFN-γ mediated induction of STAT1 phosphorylation or EGF mediated induction of ERK phosphorylation. LLL12B inhibited STAT3 phosphorylation, induced cell apoptosis, and reduced cell colony formation in triple-negative breast cancer cells. In addition, LLL12B inhibited cell migration of triple-negative breast cancer cells. Furthermore, LLL12B suppressed tumor growth of MDA-MB-231 triple-negative breast cancer cells in a mammary fat pad syngeneic mouse model. Together, our findings suggest that targeting persistent STAT3 signaling pathway by novel small molecule LLL12B may be a potential approach for triple-negative breast cancer therapy.

Citation Format: Li Pan, Xiang Chen, Feyruz V. Rassool, Chenglong Li, Jiayuh Lin. LLL12B, a novel small molecule STAT3 inhibitor induces apoptosis and suppresses cell migration and tumor growth in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2678.
Abstract 2683: Activation of MAP kinase pathway in breast cancer cell lines by polyisoprenylated cysteinyl amide inhibitors (PCAIs) causes cell death
Authors: Lazarte Jassy Mary , Tawfeeq Nada , Jin Yonghao , Lamango Nazarius
Published: June 2022

Female breast cancer has now surpassed lung cancer as the most common cancer overall in both sexes with an estimated 2.3 million new cases, representing 11.7% of all cancer cases worldwide in 2020. Triple negative breast cancer (TNBC) has been the subject of intensive studies due to its aggressive nature which makes it difficult to treat. Since inclusive treatment for this type had been elusive for decades, it is thus imperative to develop new therapeutic agents to mitigate its deadly effects. PCAIs are novel compounds designed to mimic the essential posttranslational modifications of G-proteins such as KRAS. This study elucidates the effect of the PCAIs downstream of KRAS, specifically on the mitogen activated pathway kinase (MAPK) enzymes. Two breast cancer cell lines, MDA-MB-468 and MDA-MB-231, were used to determine the levels of phosphorylation in several MAPK pathway enzymes. After exposure of the cells to varying concentrations of PCAIs (NSL-YHJ-2-27) for 48 hours, western blot results showed remarkable differences in the phosphorylated levels of the respective enzymes in cells treated with 5 µm of the PCAIs as compared to the control (NSL-YHJ-2-62). Interestingly, the cell lines reacted differently after treatment, in which MDA-MB-468 cells showed increased levels of phosphorylated MEK, ERK, and 90RSK by 122, 273 and 242%, respectively. However, in MDA-MB-231, only the phosphorylated 90RSK showed an increase of 105% and no changes in the other two enzymes. Moreover, a very distinct difference was observed in the levels of phosphorylated BRAF (p-BRAF) and CRAF (p-CRAF). In MDA-MB-231 cells, no change in p-BRAF levels while a 184% increase in p-CRAF were observed. In MDA-MB-468, p-BRAF increased by 41% and p-CRAF decreased by 58%. The differences in the response of the cell lines may reflect their unique genetics. These results show the ability of the PCAIs to interfere with the MAPK pathway. As this signaling cascade plays a very important role in cell response as well as in the progression of cancers, therefore, therapeutics targeting this pathway have a great potential in the development of treatments not only for TNBC but also for other kinds of cancers.

Citation Format: Jassy Mary Lazarte, Nada Tawfeeq, Yonghao Jin, Nazarius Lamango. Activation of MAP kinase pathway in breast cancer cell lines by polyisoprenylated cysteinyl amide inhibitors (PCAIs) causes cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2683.
Abstract 2686: Is ARV7 a regulator of prostate cancer stemness and plasticity
Authors: Furlan Tobias , Handle Florian , Cronauer Marcus V. , Santer Frédéric R.
Published: June 2022

Therapy options for advanced and castration-resistant prostate cancer (CRPC) include the use of drugs targeting directly/indirectly the ligand binding domain (LBD) of androgen receptor (AR) such as enzalutamide (Enza) and abiraterone acetate. However, primary or acquired treatment failures are frequently observed and have been associated with the expression of truncated AR variants devoid of the LBD. In particular, the AR variant AR3, also known as ARV7, is detected in over 80% of CRPC specimen and is increasingly expressed in metastatic lesions. In PC models ARV7 expression causes treatment resistance to Enza. Moreover, ARV7 expression leads to increased expression of mesenchymal markers and stem cell signature genes. CDH2 (N-Cadherin) expression is increased by ARV7 binding to regulatory elements in intron 1 of CDH2, which is antagonized by full length (FL) AR. Based on these observations we hypothesize that ARV7 is a marker and regulator of PC stemness and/or plasticity.

ARFL or ARV7 mRNA and protein was detected in cell lines and tissue samples by standard procedures including qRT-PCR, RNA in situ hybridization (RISH), immunofluorescence (IF) and western blotting using the monoclonal ARV7 antibody RM7. ARFL or ARV7 stably expressing LNCaP were obtained by lentiviral transduction. LNCaP cells resistant to Enza (LNCaP-Enza) were generated by exposure to increasing concentrations (up to 5 µM) of the drug. Transcriptome analysis in LNCaP-Enza vs. LNCaP was done by RNAseq and Gene Set Enrichment Analysis.

ARV7 mRNA was detectable in LNCaP, albeit gene expression was 2.3% - 2.6% of the known ARV7 positive cell lines 22Rv1 and VCaP, respectively. RISH and IF demonstrated that only a fraction (~10%) of LNCaP cells expressed ARV7 mRNA and nuclear localized ARV7 protein. ARV7 mRNA was also detected in isolated epithelial and stromal areas in treatment-naïve prostate cancer specimen by RISH. In contrast, all LNCaP-Enza cells were positive for nuclear ARV7 protein. Transcriptome analysis of LNCaP-Enza cells showed enrichments for published ARV7 gene expression signatures.

Insular spots of ARV7 mRNA were detected in treatment-naïve tissue and in isolated cells of the castration-sensitive cell line LNCaP suggesting that these cells could be intrinsically resistant against LBD-targeting drugs and responsible for the failure of various endocrine therapies. This project will further analyze whether this subpopulation possesses stem cell (-like)/plasticity characteristics and whether these are regulated by ARV7.

Citation Format: Tobias Furlan, Florian Handle, Marcus V. Cronauer, Frédéric R. Santer. Is ARV7 a regulator of prostate cancer stemness and plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2686.
Abstract 2698: ERα and NAMPT crosstalk at enhancers drives Fulvestrant resistance in metastatic breast tumors
Authors: Mogol Ayca N. , Chien Christine , Zuo Qianying , Raghavendra Akshara S. , Tripathy Debu , Landesman Yosef , Erdogan Zeynep Madak
Published: June 2022

Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing a potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis, with a 5-year relative overall survival rate of just 24% for patients with metastatic disease. Previously, we described an increased reliance on NAD+ metabolism in metastatic breast cancer (MBC) cells, which are driven by clonal selection from monotherapy resistance and the metastatic organ microenvironment. In the current study, we investigated if inhibition of NAMPT to block NAD+ production would synergize with standard of care therapies for ER+ MBC. Combination therapy of the NAMPT Inhibitor, KPT9274 and the ERα agonist Fulvestrant (Fulv) synergistically reduced tumor burden in liver metastasis in vitro (combination index < 1 in cell viability assays) and in vivo in mouse xenograft models. A synergistic effect was not observed when KPT-9274 was combined with Palbociclib or Tamoxifen, nor Fulv with other metabolic inhibitors. RNA-Seq analysis showed that NAMPT inhibition restored antiestrogenic activity of Fulv. Metabolomics analysis showed that NAMPT inhibition reduced abundance of metabolites associated with glucose and lipid metabolism. Mechanistically, chromatin immunoprecipitation showed that NAMPT is recruited to ERα binding sites in MBC xenografts and PDX tumors. Targeting metabolic adaptations in endocrine-resistant metastatic breast tumors is a novel approach that could lead to new combined therapies that reduce mortality.

Citation Format: Ayca N. Mogol, Christine Chien, Qianying Zuo, Akshara S. Raghavendra, Debu Tripathy, Yosef Landesman, Zeynep Madak Erdogan. ERα and NAMPT crosstalk at enhancers drives Fulvestrant resistance in metastatic breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2698.
Abstract 2701: Multivariate transcriptome analysis identifies networks and key drivers of metastatic castration-resistant prostate cancer
Authors: McKinney Lawrence P. , Singh Rajesh , Jordan I. King , Varambally Sooryanarayana , Sanda Martin G. , Dammer Eric B. , Lillard James W.
Published: June 2022

Prostate cancer (PCa) is the second most cancer-related cause of death in men. Most PCa are characterized as slow-growing or indolent, though some patients are at a high risk of for recurrent and eventually metastatic disease. The most lethal form of PCa is metastatic castration-resistant prostate cancer (mCRPC) that has progressed to the bone. The molecular mechanism underlying PCa progression to bone has not been fully elucidated. Here, we identify gene networks associated with CRPC bone metastasis and survival risk. Networks were investigated by using a novel weighted gene network co-expression analysis (WGCNA) method and examining overrepresentation of upstream regulators and signaling pathways within co-expressed transcriptome modules across clinically annotated transcriptomes from mCRPC patients (N=60). Functional Enrichment analysis was used, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction analysis, to identify biological functions of related hub genes overrepresented in our module of interest. WGCNA identified that cyan module was significantly associated with castration-resistant prostate cancer (CRPC) bone metastasis. KEGG and GO analysis results revealed genes in blue module were mainly related to collagen formation, cell signaling peptides, and bone regulation processes, including alkaline phosphatase, osteoblast formation, and endochondral ossification. Genes positively correlated with bone metastasis exhibited the following biological pathways: PI3K-Akt signaling, ECM-receptor interaction, and protein digestion and absorption pathways. This study provides novel insights into the biological pathways associated with CPRC metastasis to the bone. The module associated with bone metastasis and overall survival represent both known and novel pathways.

Citation Format: Lawrence P. McKinney, Rajesh Singh, I. King Jordan, Sooryanarayana Varambally, Martin G. Sanda, Eric B. Dammer, James W. Lillard. Multivariate transcriptome analysis identifies networks and key drivers of metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2701.
Abstract 2710: Associations between structural variant signatures and drug sensitivity in cell lines
Authors: Dalin Simona , Dubois Frank , Zhang Shu , Crane Alexander , Kalfon Jeremie , Noorbakhsh Javad , Beroukhim Rameen
Published: June 2022

Structural variants (SVs) within the cancer genome reflect defects in DNA damage repair, impact gene regulation, and alter the genomic landscape. SVs impact a larger fraction of the cancer genome than any other genetic aberration, yet their effects on modulating therapeutic response have been largely uncharacterized. Patterns of rearrangements, otherwise known as signatures, vary across tumor and tissue type, and can indicate underlying biological mechanisms responsible for cancer etiology. We have extracted SV signatures from whole genome sequencing data from hundreds of cancer cell lines. Using datasets indicating the cell lines’ response to over a thousand small molecule inhibitors, we have identified associations between SV signatures and response to therapeutics. These results will enhance our understanding of mechanisms underlying rearrangement signatures and nominate new biomarkers of drug response.

Citation Format: Simona Dalin, Frank Dubois, Shu Zhang, Alexander Crane, Jeremie Kalfon, Javad Noorbakhsh, Rameen Beroukhim. Associations between structural variant signatures and drug sensitivity in cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2710.
Abstract 2712: Establishing the early genetic progression of cancers with unobtainable premalignant disease
Authors: Leshchiner Ignaty , Mroz Edmund A. , Cha Justin , Rosebrock Daniel , Spiro Oliver , Bonilla-Velez Juliana , Faquin William , Lefranc-Torres Armida , Lin Derrick , Michaud William , Getz Gad , Rocco James W.
Published: June 2022

Premalignant tissue samples collected at different timepoints along the path to invasive tumors have been used to establish the typical order of somatic genetic events in several cancer types. For many other cancer types, however, precancer tissue is unobtainable, leaving the genetic progression unknown. Here, we demonstrate an approach that can reconstruct progression histories for such cancers from exome sequencing of primary tumors. We validated our recent computational method, PhylogicNDT, on classic, HPV-negative head and neck squamous cell carcinoma (HNSCC), recapitulating the previous experimentally determined sequence of genetic events that occur during progression. We uncovered progression of HPV-positive HNSCC tumors, which lack premalignant tissue, identifying subtype specific early drivers. We associated the timing of major changes in ploidy with the development of intra-tumor genetic heterogeneity and shorter overall survival. In cancers with unobtainable premalignant tissue, our approach can fill the knowledge gap about early progression, providing the basis for development of experimental models and methods for early detection, interception, and prognostication.

Citation Format: Ignaty Leshchiner, Edmund A. Mroz, Justin Cha, Daniel Rosebrock, Oliver Spiro, Juliana Bonilla-Velez, William Faquin, Armida Lefranc-Torres, Derrick Lin, William Michaud, Gad Getz, James W. Rocco. Establishing the early genetic progression of cancers with unobtainable premalignant disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2712.
Abstract 2731: Longitudinally modeling anti-neoantigen immunity under paradigms of immunodominance
Authors: Qi Tim , Vincent Benjamin , Cao Yanguang
Published: June 2022

Neoantigen clonal architecture is known to influence the efficacy of immunotherapy. The immunodominance of clonal neoantigens over a canvas of heterogeneous subclonal neoantigens is particularly challenging to recapitulate in experimental models. We developed a computational model of neoantigen evolution that longitudinally tracks the “presentome” - that is, a cell’s aggregate neoantigen repertoire and HLA status - of simulated non-small cell lung cancer (NSCLC) prior to and during treatment with checkpoint blockade. Using neoantigen immunogenicity parameters from published reports, we first recapitulated the presentome of early-stage NSCLC. Upon simulating checkpoint blockade treatment of advanced NSCLC, we found salient correlates and consequences of response, including a profound clonal collapse among completely responding tumors. Our work could be adapted to longitudinally model the neoantigen architecture of other cancers and modalities of immunotherapy, ultimately informing cognizant strategies for future therapeutic development.

Citation Format: Tim Qi, Benjamin Vincent, Yanguang Cao. Longitudinally modeling anti-neoantigen immunity under paradigms of immunodominance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2731.
Abstract 2755: Transcriptomic changes experienced by activated and expanded NK cells for their use in the treatment of hematological malignancies
Authors: Reina-Ortiz Chantal , Alonso Pilar Mozas , Cebollada Alberto , Ovelleiro David , Bernal Luis Alberto Anel
Published: June 2022

Natural Killer (NK) cells have shown great promise as a viable cell-based cancer immunotherapy. We have developed a protocol that produces expanded and highly activated NK (eNK) cells from healthy human donors that have been shown to be effective alone and in combination with monoclonal antibodies in a variety of hematological malignancies. The eNK cells showed a dramatic increase in cytolytic capacity after their expansion and activation using IL-2, IL-15, and 721.221 feeder cells. In order to elucidate the underlying mechanisms behind the changes, mRNAseq and miRNA arrays were performed on mRNA and miRNA samples, respectively, extracted from several NK expansions, taken at Day 0 (before activation) and Day 20 (after activation and expansion). Bioinformatic analysis was performed to identify differential gene expression changes and their downstream effects. Based on the genes with greatest differential expression, we quantified their corresponding proteins using flow cytometry, western blots, and ELISAs. Results reveal several discrete changes in the transcriptome that are reflected in changes in protein expression levels within the eNK cells. We conclude that the increased cytolytic capacity of eNK cells is due to the synergistic effect of variations in different genes responsible for target recognition, effector function, cell cycle progression and cell survival. These data can be used in the future to produce better eNK cells as treatment for hematological malignancies.

Citation Format: Chantal Reina-Ortiz, Pilar Mozas Alonso, Alberto Cebollada, David Ovelleiro, Luis Alberto Anel Bernal. Transcriptomic changes experienced by activated and expanded NK cells for their use in the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2755.
Abstract 2768: Whole-slide multispectral imaging reveals the immune subtypes of melanoma associated with the tumor microenvironment
Authors: Patel Bhavika , Allen Stephanie , Boldt Brittney , Mammadova Najiba , Haggerty Agnes , Mehra Navi
Published: June 2022

Immunotherapy and precision medicine are rapidly developing approaches to cancer therapy. Biomarkers that detect the tumor and tumor microenvironment allow for the development of strategies that accelerate the advancement of treatments to enhance a patient’s immune system. Akoya’s MOTiF™ PD-1/PD-L1 Auto Melanoma Kit is a validated, multiplex immunoassay enabling detection of the 6 most clinically relevant immuno-oncology and spatial biomarkers: FoxP3, PD-L1, Sox10/S100, PD-1, CD8 and CD68. In this study, the MOTiF™ PD-1/PD-L1 panel was used to analyze the tumor microenvironment and specifically assess immune phenotypes within melanoma samples from 3 patients. This study demonstrates a fully optimized and end-to-end workflow solution for biomarker discovery in melanoma. We demonstrate the utility of Akoya’s MOTiF™ PD-1/PD-L1 Melanoma panel kit in studying the cellular diversity while retaining spatial context. Stained slides were analyzed using the InForm® and PhenoptrReports image analysis platforms to identify phenotypes and better understand spatial relationships between cell phenotypes. The MOTiF™ PD-1/PD-L1 panel kit provides reproducibility across different patient samples. This data provides insight into the innate and adaptive immune landscape for targeted design of new immunotherapies as well as improved efficacy and reduced toxicity in the treatment of melanoma.

Citation Format: Bhavika Patel, Stephanie Allen, Brittney Boldt, Najiba Mammadova, Agnes Haggerty, Navi Mehra. Whole-slide multispectral imaging reveals the immune subtypes of melanoma associated with the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2768.
Abstract 2798: A multi-biomarker panel of nuclear transport proteins with potential as novel secreted cancer biomarkers
Authors: van der Watt Pauline , Okpara Michael , Wishart Andrew , Parker Iqbal , Soares Nelson , Blackburn Jonathan , Leaner Virna D.
Published: June 2022

Members of the nuclear transport protein family have been identified to have increased expression in many cancer types, with some being investigated as anticancer therapeutic targets. Recently, individual nuclear transport proteins have been reported to be secreted by cancer cells and found in the serum of cancer patients. In this study we investigated a multi-marker panel of nuclear transport proteins for their potential as diagnostic markers for cancer using cervical and oesophageal cancer as disease models. Mass spectrometry identified ten nuclear transport proteins in the secretome and exosomes of cultured cancer cells which was confirmed by western blot analysis showing increased secreted levels in cancer cells compared to normal. The presence of multiple nuclear transport proteins was confirmed in patient serum using ELISA assays which revealed significantly increased levels of KPNβ1, CRM1, CAS, IPO5 and TNPO1 in cervical and oesophageal cancer patient serum compared to non-cancer controls. Significantly elevated KPNα2 and RAN levels were also identified in oesophageal cancer serum samples. Logistics regression analyses revealed IPO5 and TNPO1 to be the best performing individual candidate biomarkers in discriminating between cancer cases and controls. The combination of KPNβ1, CRM1, KPNα2, CAS, RAN, IPO5 and TNPO1 as a multi-marker panel of biomarkers had the highest diagnostic capacity with an area under the curve (AUC) of 0.944 and 0.963, for cervical cancer and oesophageal cancer, and sensitivity of 92.5% at 86.8% specificity and 95.3% sensitivity at 87.5% specificity, respectively. These results suggest that nuclear transport proteins have potential as diagnostic biomarkers for cervical and oesophageal cancers, with a multi-panel protein combination being the best predictor.

Citation Format: Pauline van der Watt, Michael Okpara, Andrew Wishart, Iqbal Parker, Nelson Soares, Jonathan Blackburn, Virna D. Leaner. A multi-biomarker panel of nuclear transport proteins with potential as novel secreted cancer biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2798.
Abstract 2817: GD2 CAR-T cells engineered using retroviral transduction or CRISPR editing exhibit strong cytolytic potency against glioma stem cells
Authors: Chvatal Stacie A. , Logun Meghan T. , Sullivan Denise D. , Hayes Heather B. , Millard Daniel , Katie Mueller P. , Piscopo Nicole J. , Das Amritava , Saha Kris , Brat Daniel J. , Karumbaiah Lohitash
Published: June 2022

Glioblastoma (GBM) is an aggressive brain cancer without effective treatments. CAR-T cells targeted to tumor-associated antigens offer promise for treating GBM. Here, we used cellular impedance assays to compare the cytolytic potency and kinetics of conventional viral vs non-viral CRISPR engineered GD2 CAR-T cells against glioma stem cells (GSC), a subpopulation of glioblastoma cells.

Patient-derived N08 GSCs were plated at 50k cells/well on 96-well plates, and impedance was continuously monitored on the Maestro Z impedance platform (Axion BioSystems). GD2 CAR-T cells were engineered using either retroviral transduction (RV) or non-viral CRISPR editing (NV). At 48 hours, GD2 CAR-T cells were added at Effector:Target ratios of 0.1:1, 1:1, and 10:1. Comparisons were made to mCherry T cells (mCh) as a control. Impedance and cytolysis were monitored up to 7 days.

RV and NV GD2 CAR-T cells caused decreases in impedance consistent with T cell-mediated lysis of GSCs, whereas mCh T cells induced little change. NV CAR-T cells exhibited faster killing kinetics compared to RV CAR-T cells. The time to 50% cytolysis (KT50) was significantly shorter for NV vs RV CAR-T cells at 1:1 and 10:1 E:T ratios.

Cytotoxic function was validated with flow cytometry and cytokine analysis at 7 days. All T cells exhibited chronic activation measured by CD69 and CD137 upregulation. Importantly, NV CAR-T cells exhibited less exhaustion, as measured by PD1 and LAG3 expression.

Both RV and NV GD2 CAR-T cells effectively cytolyzed GSCs, with NV CAR-T cells exhibiting more potent and efficient killing. The high potency, fast kinetics, and reduced exhaustion of NV CRISPR GD2 CAR-T cells offer great clinical promise for treating GBM.

Citation Format: Stacie A. Chvatal, Meghan T. Logun, Denise D. Sullivan, Heather B. Hayes, Daniel Millard, Mueller P. Katie, Nicole J. Piscopo, Amritava Das, Kris Saha, Daniel J. Brat, Lohitash Karumbaiah. GD2 CAR-T cells engineered using retroviral transduction or CRISPR editing exhibit strong cytolytic potency against glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2817.