bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Protein Kinase Inhibitor H89 Enhances the Activity of Pseudomonas Exotoxin A–Based Immunotoxins
Authors: Liu Xiufen , Müller Fabian , Wayne Alan S. , Pastan Ira
Published: April 2023

Abstract
HA22 (Moxetumomab pasudotox) is a recombinant immunotoxin (RIT), composed of an anti-CD22 Fv fused to a truncated portion of Pseudomonas exotoxin A. HA22 is in clinical trials to treat patients with hairy cell leukemia and acute lymphoblastic leukemia (ALL). LMB-11 is an improved variant of HA22 with reduced immunogenicity, has a longer half-life in the blood and high activity in vitro and in a Burkitt lymphoma model in vivo. Searching for RIT enhancing combination therapies, we found the protein kinase A inhibitor H89 to enhance LMB-11 and HA22 activity 5- to 10-fold on ALL cell lines and on patient-derived ALL samples. In addition, H89 increased the activity of mesothelin-targeting RITs SS1P (38-fold) and RG7787 (7-fold) against the cervical cancer cell line KB31. Unexpectedly we found that the enhancement by H89 was not because of inhibition of protein kinase A; it was partially recapitulated by inhibition of S6K1, which led to inactivation of its downstream targets rpS6 and GSK3β, resulting in a fall in MCL1 levels. H89 increased the rate of ADP-ribosylation of eukaryotic elongation factor 2, enhancing the arrest of protein synthesis and the reduction of MCL1 in synergy with the RIT. In summary, H89 increased RIT activity by enhancing the two key events: ADP-ribosylation of eEF2 and reduction of MCL1 levels. Significant enhancement was seen with both CD22- and mesothelin-targeting RITs, indicating that H89 might be a potent addition to RIT treatment of CD22-positive ALL and mesothelin-expressing solid tumors. Mol Cancer Ther; 15(5); 1053–62. ©2016 AACR.
Data from Combination Therapy with c-Met and Src Inhibitors Induces Caspase-Dependent Apoptosis of Merlin-Deficient Schwann Cells and Suppresses Growth of Schwannoma Cells
Authors: Fuse Marisa A. , Plati Stephani Klingeman , Burns Sarah S. , Dinh Christine T. , Bracho Olena , Yan Denise , Mittal Rahul , Shen Rulong , Soulakova Julia N. , Copik Alicja J. , Liu Xue Zhong , Telischi Fred F. , Chang Long-Sheng , Franco Maria Clara , Fernandez-Valle Cristina
Published: April 2023

Abstract
Neurofibromatosis type 2 (NF2) is a nervous system tumor disorder caused by inactivation of the merlin tumor suppressor encoded by the NF2 gene. Bilateral vestibular schwannomas are a diagnostic hallmark of NF2. Mainstream treatment options for NF2-associated tumors have been limited to surgery and radiotherapy; however, off-label uses of targeted molecular therapies are becoming increasingly common. Here, we investigated drugs targeting two kinases activated in NF2-associated schwannomas, c-Met and Src. We demonstrated that merlin-deficient mouse Schwann cells (MD-MSC) treated with the c-Met inhibitor, cabozantinib, or the Src kinase inhibitors, dasatinib and saracatinib, underwent a G1 cell-cycle arrest. However, when MD-MSCs were treated with a combination of cabozantinib and saracatinib, they exhibited caspase-dependent apoptosis. The combination therapy also significantly reduced growth of MD-MSCs in an orthotopic allograft mouse model by greater than 80% of vehicle. Moreover, human vestibular schwannoma cells with NF2 mutations had a 40% decrease in cell viability when treated with cabozantinib and saracatinib together compared with the vehicle control. This study demonstrates that simultaneous inhibition of c-Met and Src signaling in MD-MSCs triggers apoptosis and reveals vulnerable pathways that could be exploited to develop NF2 therapies. Mol Cancer Ther; 16(11); 2387–98. ©2017 AACR.
Data from A Novel Glycogen Synthase Kinase-3 Inhibitor Optimized for Acute Myeloid Leukemia Differentiation Activity
Authors: Hu Sophia , Ueda Masumi , Stetson Lindsay , Ignatz-Hoover James , Moreton Stephen , Chakrabarti Amit , Xia Zhiqiang , Karan Goutam , de Lima Marcos , Agrawal Mukesh K. , Wald David N.
Published: April 2023

Abstract
Standard therapies used for the treatment of acute myeloid leukemia (AML) are cytotoxic agents that target rapidly proliferating cells. Unfortunately, this therapeutic approach has limited efficacy and significant toxicity and the majority of AML patients still die of their disease. In contrast to the poor prognosis of most AML patients, most individuals with a rare subtype of AML, acute promyelocytic leukemia, can be cured by differentiation therapy using regimens containing all-trans retinoic acid. GSK3 has been previously identified as a therapeutic target in AML where its inhibition can lead to the differentiation and growth arrest of leukemic cells. Unfortunately, existing GSK3 inhibitors lead to suboptimal differentiation activity making them less useful as clinical AML differentiation agents. Here, we describe the discovery of a novel GSK3 inhibitor, GS87. GS87 was discovered in efforts to optimize GSK3 inhibition for AML differentiation activity. Despite GS87's dramatic ability to induce AML differentiation, kinase profiling reveals its high specificity in targeting GSK3 as compared with other kinases. GS87 demonstrates high efficacy in a mouse AML model system and unlike current AML therapeutics, exhibits little effect on normal bone marrow cells. GS87 induces potent differentiation by more effectively activating GSK3-dependent signaling components including MAPK signaling as compared with other GSK3 inhibitors. GS87 is a novel GSK3 inhibitor with therapeutic potential as a differentiation agent for non-promyelocytic AML. Mol Cancer Ther; 15(7); 1485–94. ©2016 AACR.
Data from Reactivation of p53 by MDM2 Inhibitor MI-77301 for the Treatment of Endocrine-Resistant Breast Cancer
Authors: Lu Jianfeng , McEachern Donna , Li Shunqiang , Ellis Matthew J. , Wang Shaomeng
Published: April 2023

Abstract
Endocrine therapy has been highly effective for the treatment of estrogen receptor–positive breast cancer, but endocrine resistance develops in a significant proportion of patients. In an effort to develop novel therapeutic strategies for the treatment of endocrine-resistant breast cancer, we have evaluated a potent and specific MDM2–p53 interaction inhibitor, MI-77301, which has been advanced into clinical development, for its therapeutic potential and mechanism of action in vitro and in vivo in WHIM9 and WHIM18 patient-derived xenograft (PDX) models. Both WHIM9 and WHIM18 PDX models exhibit estradiol-independent tumor growth and are resistant to fulvestrant, a highly effective and selective estrogen receptor degrader (SERD). MI-77301 activates wild-type p53 in WHIM9 and WHIM18 cells in vitro and in xenograft tumor tissues in vivo, and it effectively induces upregulation of p21 and cell-cycle arrest in vitro in both models. Although fulvestrant fails to inhibit tumor growth in either of the xenograft models, MI-77301 is highly effective in inhibition of tumor growth at a well-tolerated dose schedule. This study provides a preclinical rationale for evaluation of MI-77301 or other MDM2 inhibitors as a new therapeutic strategy for the treatment of endocrine-resistant breast cancer retaining wild-type p53. Mol Cancer Ther; 15(12); 2887–93. ©2016 AACR.
Data from Autophagy Inhibition Improves Sunitinib Efficacy in Pancreatic Neuroendocrine Tumors via a Lysosome-dependent Mechanism
Authors: Wiedmer Tabea , Blank Annika , Pantasis Sophia , Normand Lea , Bill Ruben , Krebs Philippe , Tschan Mario P. , Marinoni Ilaria , Perren Aurel
Published: April 2023

Abstract
Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine tumors (PanNET) is an unmet medical need. The antiangiogenic tyrosine kinase inhibitor sunitinib is approved for PanNET treatment. In addition, sunitinib is a lysosomotropic drug and such drugs can induce lysosomal membrane permeabilization as well as autophagy. We investigated sunitinib-induced autophagy as a possible mechanism of PanNET therapy resistance. Sunitinib accumulated in lysosomes and induced autophagy in PanNET cell lines. Adding the autophagy inhibitor chloroquine reduced cell viability in cell lines and in primary cells isolated from PanNET patients. The same treatment combination reduced tumor burden in the Rip1Tag2 transgenic PanNET mouse model. The combination of sunitinib and chloroquine reduced recovery and induced apoptosis in vitro, whereas single treatments did not. Knockdown of key autophagy proteins in combination with sunitinib showed similar effect as chloroquine. Sunitinib also induced lysosomal membrane permeabilization, which further increased in the presence of chloroquine or knockdown of lysosome-associated membrane protein (LAMP2). Both combinations led to cell death. Our data indicate that chloroquine increases sunitinib efficacy in PanNET treatment via autophagy inhibition and lysosomal membrane permeabilization. We suggest that adding chloroquine to sunitinib treatment will increase efficacy of PanNET treatment and that such patients should be included in respective ongoing clinical trials. Mol Cancer Ther; 16(11); 2502–15. ©2017 AACR.
Data from Paclitaxel-Loaded Polymersomes for Enhanced Intraperitoneal Chemotherapy
Authors: Simón-Gracia Lorena , Hunt Hedi , Scodeller Pablo D. , Gaitzsch Jens , Braun Gary B. , Willmore Anne-Mari A. , Ruoslahti Erkki , Battaglia Giuseppe , Teesalu Tambet
Published: April 2023

Abstract
Peritoneal carcinomatosis is present in more than 60% of gastric cancer, 40% of ovarian cancer, and 35% of colon cancer patients. It is the second most common cause of cancer-related mortality, with a median survival of 1 to 3 months. Cytoreductive surgery combined with intraperitoneal chemotherapy is the current clinical treatment, but achieving curative drug accumulation and penetration in peritoneal carcinomatosis lesions remains an unresolved challenge. Here, we used flexible and pH-sensitive polymersomes for payload delivery to peritoneal gastric (MKN-45P) and colon (CT26) carcinoma in mice. Polymersomes were loaded with paclitaxel and in vitro drug release was studied as a function of pH and time. Paclitaxel-loaded polymersomes remained stable in aqueous solution at neutral pH for up to 4 months. In cell viability assay on cultured cancer cell lines (MKN-45P, SKOV3, CT26), paclitaxel-loaded polymersomes were more toxic than free drug or albumin-bound paclitaxel (Abraxane). Intraperitoneally administered fluorescent polymersomes accumulated in malignant lesions, and immunofluorescence revealed an intense signal inside tumors with no detectable signal in control organs. A dual targeting of tumors was observed: direct (circulation-independent) penetration, and systemic, blood vessel–associated accumulation. Finally, we evaluated preclinical antitumor efficacy of paclitaxel-polymersomes in the treatment of MKN-45P disseminated gastric carcinoma using a total dose of 7 mg/kg. Experimental therapy with paclitaxel-polymersomes improved the therapeutic index of drug over free paclitaxel and Abraxane, as evaluated by intraperitoneal tumor burden and number of metastatic nodules. Our findings underline the potential utility of the polymersome platform for delivery of drugs and imaging agents to peritoneal carcinomatosis lesions. Mol Cancer Ther; 15(4); 670–9. ©2016 AACR.
Data from Akt Activation Mediates Acquired Resistance to Fibroblast Growth Factor Receptor Inhibitor BGJ398
Authors: Datta Jharna , Damodaran Senthilkumar , Parks Hannah , Ocrainiciuc Cristina , Miya Jharna , Yu Lianbo , Gardner Elijah P. , Samorodnitsky Eric , Wing Michele R. , Bhatt Darshna , Hays John , Reeser Julie W. , Roychowdhury Sameek
Published: April 2023

Abstract
Activation of FGFR signaling through mutations, amplifications, or fusions involving FGFR1, 2, 3, or 4 is seen in multiple tumors, including lung, bladder, and cholangiocarcinoma. Currently, several clinical trials are evaluating the role of novel FGFR inhibitors in solid tumors. As we move forward with FGFR inhibitors clinically, we anticipate the emergence of resistance with treatment. Consequently, we sought to study the mechanism(s) of acquired resistance to FGFR inhibitors using annotated cancer cell lines. We identified cancer cell lines that have activating mutations in FGFR1, 2, or 3 and treated them chronically with the selective FGFR inhibitor, BGJ398. We observed resistance to chronic BGJ398 exposure in DMS114 (small-cell lung cancer, FGFR1 amplification) and RT112 (urothelial carcinoma, FGFR3 fusion/amplification) cell lines based on viability assays. Reverse-phase protein array (RPPA) analysis showed increased phosphorylation of Akt (T308 and S473) and its downstream target GSK3 (S9 and S21) in both the resistant cell lines when compared with matching controls. Results of RPPA were confirmed using immunoblots. Consequently, the addition of an Akt inhibitor (GSK2141795) or siRNA was able to restore sensitivity to BGJ398 in resistant cell lines. These data suggest a role for Akt pathway in mediating acquired resistance to FGFR inhibition. Mol Cancer Ther; 16(4); 614–24. ©2017 AACR.
Data from Genome-Wide Sequencing of Cell-Free DNA Identifies Copy-Number Alterations That Can Be Used for Monitoring Response to Immunotherapy in Cancer Patients
Authors: Jensen Taylor J. , Goodman Aaron M. , Kato Shumei , Ellison Christopher K. , Daniels Gregory A. , Kim Lisa , Nakashe Prachi , McCarthy Erin , Mazloom Amin R. , McLennan Graham , Grosu Daniel S. , Ehrich Mathias , Kurzrock Razelle
Published: April 2023

Abstract
Inhibitors of the PD-1/PD-L1/CTLA-4 immune checkpoint pathway have revolutionized cancer treatment. Indeed, some patients with advanced, refractory malignancies achieve durable responses; however, only a subset of patients benefit, necessitating new biomarkers to predict outcome. Interrogating cell-free DNA (cfDNA) isolated from plasma (liquid biopsy) provides a promising method for monitoring response. We describe the use of low-coverage, genome-wide sequencing of cfDNA, validated extensively for noninvasive prenatal testing, to detect tumor-specific copy-number alterations, and the development of a new metric—the genome instability number (GIN)—to monitor response to these drugs. We demonstrate how the GIN can be used to discriminate clinical response from progression, differentiate progression from pseudoprogression, and identify hyperprogressive disease. Finally, we provide evidence for delayed kinetics in responses to checkpoint inhibitors relative to molecularly targeted therapies. Overall, these data demonstrate a proof of concept for using this method for monitoring treatment outcome in patients with cancer receiving immunotherapy.
Data from Ascochlorin Enhances the Sensitivity of Doxorubicin Leading to the Reversal of Epithelial-to-Mesenchymal Transition in Hepatocellular Carcinoma
Authors: Dai Xiaoyun , Ahn Kwang Seok , Wang Ling Zhi , Kim Chulwon , Deivasigamni Amudha , Arfuso Frank , Um Jae-Young , Kumar Alan Prem , Chang Young-Chae , Kumar Dhiraj , Kundu Gopal C. , Magae Junji , Goh Boon Cher , Hui Kam Man , Sethi Gautam
Published: April 2023

Abstract
Increasing evidence has indicated that epithelial-to-mesenchymal transition (EMT) at the advanced stage of liver cancer not only has the ability to self-renew and progress cancer, but also enables greater resistance to conventional chemo- and radiotherapies. Here, we report that ascochlorin (ASC), an isoprenoid antibiotic, could potentiate the cytotoxic effect of doxorubicin on HCCLM3, SNU387, SNU49, and SK-Hep-1 hepatocellular carcinoma cells, which had a predominantly mesenchymal signature with low expression of E-cadherin but high expression of N-cadherin. Co-administration of ASC reduced doxorubicin-induced invasion/migration and modulated EMT characteristics in mesenchymal cells. This process was probably mediated by the E-cadherin repressors Snail and Slug. In addition, ASC increased sensitivity to doxorubicin treatment by directly inhibiting STAT3 binding to the Snail promoter. We also observed that ASC significantly enhanced the effect of doxorubicin against tumor growth and inhibited metastasis in an HCCLM3_Luc orthotopic mouse model. Collectively, our data demonstrate that ASC can increase sensitivity to doxorubicin therapy and reverse the EMT phenotype via the downregulation of STAT3-Snail expression, which could form the basis of a novel therapeutic approach against hepatocellular carcinoma. Mol Cancer Ther; 15(12); 2966–76. ©2016 AACR.
Data from Bispecific Antibodies and Antibody–Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs
Authors: Andreev Julian , Thambi Nithya , Perez Bay Andres E. , Delfino Frank , Martin Joel , Kelly Marcus P. , Kirshner Jessica R. , Rafique Ashique , Kunz Arthur , Nittoli Thomas , MacDonald Douglas , Daly Christopher , Olson William , Thurston Gavin
Published: April 2023

Abstract
The properties of cell surface proteins targeted by antibody–drug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR (∼30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 (∼106 surface receptors per cell). Noncovalently cross-linking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs. Mol Cancer Ther; 16(4); 681–93. ©2017 AACR.
Data from Aurora A Inhibitor TAS-119 Enhances Antitumor Efficacy of Taxanes In Vitro and In Vivo: Preclinical Studies as Guidance for Clinical Development and Trial Design
Authors: Sootome Hiroshi , Miura Akihiro , Masuko Norio , Suzuki Takamasa , Uto Yoshihiro , Hirai Hiroshi
Published: April 2023

Abstract
TAS-119 is a novel orally active, selective inhibitor of Aurora kinase A identified as a clinical candidate for efficacy testing in combination with taxanes. In vitro, TAS-119 enhanced cell growth inhibition of paclitaxel in multiple human cancer cell lines derived from various tissues, including paclitaxel-resistant cell lines. Interestingly, TAS-119 did not enhance paclitaxel antitumor activity in normal lung diploid fibroblast cell lines WI-38 and MRC5. In vivo, TAS-119 enhanced the antitumor efficacy of paclitaxel and docetaxel in multiple models at doses inhibitory to Aurora A in tumors. Moreover, the drug combination was well tolerated, and TAS-119 did not exaggerate clinically documented side effects of taxanes, neutropenia and neurotoxicity, in rats. The same TAS-119 concentration enhanced the cell growth inhibitory activity of three clinically approved taxanes, paclitaxel, docetaxel, and cabazitaxel. The degree of enhancement calculated as fold of change of the IC50 value for each taxane was almost the same among the three taxanes. We conducted in vitro and in vivo experiments to develop an optimized combination therapy regimen for TAS-119 with paclitaxel/docetaxel. Using in vitro and in vivo models, we tested the drug administration order for TAS-119 combined with paclitaxel and the TAS-119 treatment duration. The best regimen in preclinical models was combining paclitaxel or docetaxel treatment with 4 days of TAS-119 dosing, which was initiated on the same day as the paclitaxel or docetaxel administration or one day later. This information provided guidance for the design of a clinical trial of TAS-119 and paclitaxel or docetaxel combination.
Data from Osimertinib, an Irreversible Next-Generation EGFR Tyrosine Kinase Inhibitor, Exerts Antitumor Activity in Various Preclinical NSCLC Models Harboring the Uncommon EGFR Mutations G719X or L861Q or S768I
Authors: Floc'h Nicolas , Lim Sangbin , Bickerton Sue , Ahmed Afshan , Orme Jonathan , Urosevic Jelena , Martin Matthew J. , Cross Darren A.E. , Cho Byoung Chul , Smith Paul D.
Published: April 2023

Abstract
Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI–sensitizing and EGFR T790M–resistance mutations with lower activity against wild-type EGFR and has demonstrated efficacy in non–small cell lung cancer (NSCLC) CNS metastases. The sensitizing mutations, the in-frame deletions in exon 19 and the L858R point mutation in exon 21, represent between 80% and 90% of all EGFR mutations. The remaining 10% to 20% are referred to as uncommon activating mutations and are a diverse group of mutations in exons 18 to 21 within the kinase domain of the EGFR gene. Excluding those found as insertion mutations in exon 20, the uncommon mutations involving codons G719, S768, and L861 are the most prevalent.

Although the efficacy of EGFR-TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations, such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series.

Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDXs that harbor the G719X mutation, we have evaluated in vitro and in vivo the preclinical activity of osimertinib. We report osimertinib inhibits signaling pathways and cellular growth in G719X-mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of PDX harboring the G719X mutation alone or in combination with L861Q and S768I.

Together, these data support clinical testing of osimertinib in patients with uncommon EGFR NSCLC.
Data from Nf1-Mutant Tumors Undergo Transcriptome and Kinome Remodeling after Inhibition of either mTOR or MEK
Authors: Pucciarelli Daniela , Angus Steven P. , Huang Benjamin , Zhang Chi , Nakaoka Hiroki J. , Krishnamurthi Ganesh , Bandyopadhyay Sourav , Clapp D. Wade , Shannon Kevin , Johnson Gary L. , Nakamura Jean L.
Published: April 2023

Abstract
Loss of the tumor suppressor NF1 leads to activation of RAS effector pathways, which are therapeutically targeted by inhibition of mTOR (mTORi) or MEK (MEKi). However, therapeutic inhibition of RAS effectors leads to the development of drug resistance and ultimately disease progression. To investigate molecular signatures in the context of NF1 loss and subsequent acquired drug resistance, we analyzed the exomes, transcriptomes, and kinomes of Nf1-mutant mouse tumor cell lines and derivatives of these lines that acquired resistance to either MEKi or mTORi. Biochemical comparisons of this unique panel of tumor cells, all of which arose in Nf1+/− mice, indicate that loss of heterozygosity of Nf1 as an initial genetic event does not confer a common biochemical signature or response to kinase inhibition. Although acquired drug resistance by Nf1-mutant tumor cells was accompanied by altered kinomes and irreversibly altered transcriptomes, functionally in multiple Nf1-mutant tumor cell lines, MEKi resistance was a stable phenotype, in contrast to mTORi resistance, which was reversible. Collectively, these findings demonstrate that Nf1-mutant tumors represent a heterogeneous group biochemically and undergo broader remodeling of kinome activity and gene expression in response to targeted kinase inhibition.
Data from Enhanced Immunotherapy with LHRH-R Targeted Lytic Peptide in Ovarian Cancer
Authors: Kim Mark Seungwook , Ma Shaolin , Chelariu-Raicu Anca , Leuschner Carola , Alila Hector W. , Lee Sanghoon , Coleman Robert L. , Sood Anil K.
Published: April 2023

Abstract
Here, we examined the role of EP-100 [luteinizing hormone-releasing hormone (LHRH) ligand joined to a lytic peptide], improving the efficacy of immune checkpoint blockade. LHRH-R–positive murine ovarian cancer cells (ID8, IG10, IF5, and 2C12) were sensitive to EP-100 and were specifically killed at low micromolar levels through LHRH-R. EP-100 increased PD-L1 levels on murine ovarian cancer cells. In vivo syngeneic mouse models (ID8 and IG10) demonstrated that single-agent EP-100 reduced tumor volume, tumor weight, and ascites volume. The greatest reductions in tumor and ascites volume were observed with the combination of EP-100 with an anti–PD-L1 antibody. Immune profiling analysis showed that the population of CD8+ T cells, natural killer cells, dendritic cells, and macrophages were significantly increased in tumor and ascitic fluid samples treated with anti–PD-L1, EP-100, and the combination. However, monocytic myeloid suppressor cells, B cells, and regulatory T cells were decreased in tumors treated with anti–PD-L1, EP-100, or the combination. In vitro cytokine arrays revealed that EP-100 induced IL1α, IL33, CCL20, VEGF, and Low-density lipoprotein receptor (LDLR) secretion. Of these, we validated increasing IL33 levels following EP-100 treatment in vitro and in vivo; we determined the specific biological role of CD8+ T-cell activation with IL33 gene silencing using siRNA and Cas9-CRISPR approaches. In addition, we found that CD8+ T cells expressed very low level of LHRH-R and were not affected by EP-100. Taken together, EP-100 treatment had a substantial antitumor efficacy, particularly in combination with an anti–PD-L1 antibody. These results warrant further clinical development of this combination.
Data from PARP1 Inhibition Radiosensitizes Models of Inflammatory Breast Cancer to Ionizing Radiation
Authors: Michmerhuizen Anna R. , Pesch Andrea M. , Moubadder Leah , Chandler Benjamin C. , Wilder-Romans Kari , Cameron Meleah , Olsen Eric , Thomas Dafydd G. , Zhang Amanda , Hirsh Nicole , Ritter Cassandra L. , Liu Meilan , Nyati Shyam , Pierce Lori J. , Jagsi Reshma , Speers Corey
Published: April 2023

Abstract
Sustained locoregional control of disease is a significant issue in patients with inflammatory breast cancer (IBC), with local control rates of 80% or less at 5 years. Given the unsatisfactory outcomes for these patients, there is a clear need for intensification of local therapy, including radiation. Inhibition of the DNA repair protein PARP1 has had little efficacy as a single agent in breast cancer outside of studies restricted to patients with BRCA mutations; however, PARP1 inhibition (PARPi) may lead to the radiosensitization of aggressive tumor types. Thus, this study investigates inhibition of PARP1 as a novel and promising radiosensitization strategy in IBC. In multiple existing IBC models (SUM-149, SUM-190, MDA-IBC-3), PARPi (AZD2281-olaparib and ABT-888-veliparib) had limited single-agent efficacy (IC50 > 10 μmol/L) in proliferation assays. Despite limited single-agent efficacy, submicromolar concentrations of AZD2281 in combination with RT led to significant radiosensitization (rER 1.12–1.76). This effect was partially dependent on BRCA1 mutational status. Radiosensitization was due, at least in part, to delayed resolution of double strand DNA breaks as measured by multiple assays. Using a SUM-190 xenograft model in vivo, the combination of PARPi and RT significantly delays tumor doubling and tripling times compared with PARPi or RT alone with limited toxicity. This study demonstrates that PARPi improves the effectiveness of radiotherapy in IBC models and provides the preclinical rationale for the opening phase II randomized trial of RT ± PARPi in women with IBC (SWOG 1706, NCT03598257).
Data from Inhibition of Lysosomal Function Mitigates Protective Mitophagy and Augments Ceramide Nanoliposome–Induced Cell Death in Head and Neck Squamous Cell Carcinoma
Authors: Shaw Jeremy J.P. , Boyer Timothy L. , Venner Emily , Beck Patrick J. , Slamowitz Tristen , Caste Tara , Hickman Alexandra , Raymond Michael H. , Costa-Pinheiro Pedro , Jameson Mark J. , Fox Todd E. , Kester Mark
Published: April 2023

Abstract
Therapies for head and neck squamous cell carcinoma (HNSCC) are, at best, moderately effective, underscoring the need for new therapeutic strategies. Ceramide treatment leads to cell death as a consequence of mitochondrial damage by generating oxidative stress and causing mitochondrial permeability. However, HNSCC cells are able to resist cell death through mitochondria repair via mitophagy. Through the use of the C6-ceramide nanoliposome (CNL) to deliver therapeutic levels of bioactive ceramide, we demonstrate that the effects of CNL are mitigated in drug-resistant HNSCC via an autophagic/mitophagic response. We also demonstrate that inhibitors of lysosomal function, including chloroquine (CQ), significantly augment CNL-induced death in HNSCC cell lines. Mechanistically, the combination of CQ and CNL results in dysfunctional lysosomal processing of damaged mitochondria. We further demonstrate that exogenous addition of methyl pyruvate rescues cells from CNL + CQ–dependent cell death by restoring mitochondrial functionality via the reduction of CNL- and CQ-induced generation of reactive oxygen species and mitochondria permeability. Taken together, inhibition of late-stage protective autophagy/mitophagy augments the efficacy of CNL through preventing mitochondrial repair. Moreover, the combination of inhibitors of lysosomal function with CNL may provide an efficacious treatment modality for HNSCC.
Data from CDK9 Blockade Exploits Context-dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma
Authors: Flores Guillermo , Everett Joel H. , Boguslawski Elissa A. , Oswald Brandon M. , Madaj Zachary B. , Beddows Ian , Dikalov Sergey , Adams Marie , Klumpp-Thomas Carleen A. , Kitchen-Goosen Susan M. , Martin Scott E. , Caplen Natasha J. , Helman Lee J. , Grohar Patrick J.
Published: April 2023

Abstract
There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor–targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1–targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic “class” effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1–targeted combination therapy for Ewing sarcoma.
Data from Efficacy, Tolerability, and Pharmacokinetics of Combined Targeted MEK and Dual mTORC1/2 Inhibition in a Preclinical Model of Mucosal Melanoma
Authors: Wei Bih-Rong , Hoover Shelley B. , Peer Cody J. , Dwyer Jennifer E. , Adissu Hibret A. , Shankarappa Priya , Yang Howard , Lee Maxwell , Peat Tyler J. , Figg William D. , Simpson R. Mark
Published: April 2023

Abstract
Melanomas arising in the mucous membranes are a rare and aggressive subtype. New treatment approaches are needed, yet accumulating sufficient evidence to improve patient outcomes is difficult. Clinical and pathological correlates between human and canine mucosal melanomas are substantial, and the relatively greater incidence of spontaneous naturally occurring mucosal melanoma in dogs represents a promising opportunity for predictive modeling. The genomic landscapes of human and canine mucosal melanoma appear highly diverse and generally lack recurring hotspot mutations associated with cutaneous melanomas. Although much remains to be determined, evidence indicates that Ras/MAPK and/or PI3K/AKT/mTOR signaling pathway activations are common in both species and may represent targets for therapeutic intervention. Sapanisertib, an mTORC1/2 inhibitor, was selected from a PI3K/mTOR inhibitor library to collaborate with MEK inhibition; the latter preclinical efficacy was demonstrated previously for canine mucosal melanoma. Combined inhibition of MEK and mTORC1/2, using trametinib and sapanisertib, produced apoptosis and cell-cycle alteration, synergistically reducing cell survival in canine mucosal melanoma cell lines with varying basal signaling activation levels. Compared with individual inhibitors, a staggered sapanisertib dose, coupled with daily trametinib, was optimal for limiting primary mucosal melanoma xenograft growth in mice, and tumor dissemination in a metastasis model, while minimizing hematologic and renal side effects. Inhibitors downmodulated respective signaling targets and the combination additionally suppressed pathway reciprocal crosstalk. The combination did not significantly change plasma sapanisertib pharmacokinetics; however, trametinib area under the curve was increased in the presence of sapanisertib. Targeting Ras/MAPK and PI3K/AKT/mTOR signal transduction pathways appear rational therapies for canine and human mucosal melanoma.
Data from Functional miRNA Screening Identifies Wide-ranging Antitumor Properties of miR-3622b-5p and Reveals a New Therapeutic Combination Strategy in Ovarian Tumor Organoids
Authors: Vernon Mégane , Lambert Bernard , Meryet-Figuière Matthieu , Brotin Emilie , Weiswald Louis-Bastien , Paysant Hippolyte , Vigneron Nicolas , Wambecke Anaïs , Abeilard Edwige , Giffard Florence , Louis Marie-Hélène , Blanc-Fournier Cécile , Gauduchon Pascal , Poulain Laurent , Denoyelle Christophe
Published: April 2023

Abstract
Novel therapeutic strategies are urgently required for the clinical management of chemoresistant ovarian carcinoma, which is the most lethal of the gynecologic malignancies. miRNAs hold promise because they play a critical role in determining the cell phenotype by regulating several hundreds of targets, which could constitute vulnerabilities of cancer cells. A combination of gain-of-function miRNA screening and real-time continuous cell monitoring allows the identification of miRNAs with robust cytotoxic effects in chemoresistant ovarian cancer cells. Focusing on miR-3622b-5p, we show that it induces apoptosis in several ovarian cancer cell lines by both directly targeting Bcl-xL and EGFR-mediating BIM upregulation. miR-3622b-5p also sensitizes cells to cisplatin by inhibiting Bcl-xL in ovarian cancer cell lines escaping BIM induction. miR-3622b-5p also exerts antimigratory capacities by targeting both LIMK1 and NOTCH1. These wide-ranging antitumor properties of miR-3622b-5p in ovarian cancer cells are mimicked by the associations of pharmacologic inhibitors targeting these proteins. The combination of an EGFR inhibitor together with a BH3-mimetic molecule induced a large decrease in cell viability in a panel of ovarian cancer cell lines and several ovarian patient-derived tumor organoids, suggesting the value of pursuing such a combination therapy in ovarian carcinoma. Altogether, our work highlights the potential of phenotype-based miRNA screening approaches to identify lethal interactions which might lead to new drug combinations and clinically applicable strategies.
Data from Genomic and Expression Analyses Define MUC17 and PCNX1 as Predictors of Chemotherapy Response in Breast Cancer
Authors: Al Amri Waleed S. , Allinson Lisa M. , Baxter Diana E. , Bell Sandra M. , Hanby Andrew M. , Jones Stacey J. , Shaaban Abeer M. , Stead Lucy F. , Verghese Eldo T. , Hughes Thomas A.
Published: April 2023

Abstract
Poor-prognosis breast cancers are treated with cytotoxic chemotherapy, but often without any guidance from therapy predictive markers because universally accepted markers are not currently available. Treatment failure, in the form of recurrences, is relatively common. We aimed to identify chemotherapy predictive markers and resistance pathways in breast cancer. Our hypothesis was that tumor cells remaining after neoadjuvant chemotherapy (NAC) contain somatic variants causing therapy resistance, while variants present pre-NAC but lost post-NAC cause sensitivity. Whole-exome sequencing was performed on matched pre- and post-NAC cancer cells, which were isolated by laser microdissection, from 6 cancer cases, and somatic variants selected for or against by NAC were identified. Somatic variant diversity was significantly reduced after therapy (P < 0.05). MUC17 variants were identified in 3 tumors and were selected against by NAC in each case, while PCNX1 variants were identified in 2 tumors and were selected for in both cases, implicating the function of these genes in defining chemoresponse. In vitro knockdown of MUC17 or PCNX1 was associated with significantly increased or decreased chemotherapy sensitivity, respectively (P < 0.05), further supporting their roles in chemotherapy response. Expression was tested for predictive value in two independent cohorts of chemotherapy-treated breast cancers (n = 53, n = 303). Kaplan–Meier analyses revealed that low MUC17 expression was significantly associated with longer survival after chemotherapy, whereas low PCNX1 was significantly associated with reduced survival. We concluded that therapy-driven selection of somatic variants allows identification of chemotherapy response genes. With respect to MUC17 and PCNX1, therapy-driven selection acting on somatic variants, in vitro knockdown data concerning drug sensitivity, and survival analysis of expression levels in patient cohorts all define the genes as mediators of and predictive markers for chemotherapy response in breast cancer.
Data from Therapy of Myeloid Leukemia using Novel Bispecific Fusion Proteins Targeting CD45 and 90Y-DOTA
Authors: Orozco Johnnie J. , Kenoyer Aimee L. , Lin Yukang , O'Steen Shyril , Guel Rosario , Nartea Margaret E. , Hernandez Alexandra H. , Hylarides Mark D. , Fisher Darrell R. , Balkin Ethan R. , Hamlin Donald K. , Wilbur D. Scott , Orcutt Kelly D. , Wittrup K. Dane , Green Damian J. , Gopal Ajay K. , Till Brian G. , Sandmaier Brenda , Press Oliver W. , Pagel John M.
Published: April 2023

Abstract
Pretargeted radioimmunotherapy (PRIT) has been investigated as a multi-step approach to decrease relapse and toxicity for high-risk acute myeloid leukemia (AML). Relevant factors including endogenous biotin and immunogenicity, however, have limited the use of PRIT with an anti-CD45 antibody streptavidin conjugate and radiolabeled DOTA-biotin. To overcome these limitations we designed anti-murine and anti-human CD45 bispecific antibody constructs using 30F11 and BC8 antibodies, respectively, combined with an anti-yttrium (Y)-DOTA single-chain variable fragment (C825) to capture a radiolabeled ligand. The bispecific construct targeting human CD45 (BC8-Fc-C825) had high uptake in leukemia HEL xenografts [7.8 ± 0.02% percent injected dose/gram of tissue (% ID/g)]. Therapy studies showed that 70% of mice with HEL human xenografts treated with BC8-Fc-C825 followed by 44.4 MBq (1,200 μCi) of 90Y-DOTA-biotin survived at least 170 days after therapy, while all nontreated controls required euthanasia because of tumor progression by day 32. High uptake at sites of leukemia (spleen and bone marrow) was also seen with 30F11-IgG1-C825 in a syngeneic disseminated SJL murine leukemia model (spleen, 9.0 ± 1.5% ID/g and bone marrow, 8.1 ± 1.2% ID/g), with minimal uptake in all other normal organs (<0.5% ID/g) at 24 hours after 90Y-DOTA injections. SJL leukemia mice treated with the bispecific 30F11-IgG1-C825 and 29.6 MBq (800 μCi) of 90Y-DOTA-biotin had a survival advantage compared with untreated leukemic mice (median, 43 vs. 30 days, respectively; P < 0.0001). These data suggest bispecific antibody–mediated PRIT may be highly effective for leukemia therapy and translation to human studies.
Data from High-Throughput Generation of Bispecific Binding Proteins by Sortase A–Mediated Coupling for Direct Functional Screening in Cell Culture
Authors: Andres Fabio , Schwill Martin , Boersma Ykelien L. , Plückthun Andreas
Published: April 2023

Abstract
High-throughput construction of multivalent binders and subsequent screening for biological activity represent a fundamental challenge: A linear increase of monovalent components translates to the square of possible bivalent combinations. Even high-efficiency cloning and expression methods become limiting when thousands of bispecific binders need to be screened for activity. In this study, we present an in vitro method for the efficient production of flexibly linked bispecific binding agents from individually expressed and purified monovalent binders. We established a sortase A–mediated coupling reaction to generate bispecific designed ankyrin repeat proteins (DARPins), with an optimized reaction maximizing the bivalent coupling product with low levels of monovalent side-products. These one-pot reaction mixtures could be used directly, without further purification, in cell-based assays. We generated a matrix of 441 different bispecific DARPins against the extracellular domains of the cancer-associated receptors EGFR, ErbB2, ErbB3, ErbB4, EpCAM, and c-MET and screened on two different ErbB2-positive cancer cells lines for growth-inhibitory effects. We identified not only known but also novel biologically active biparatopic DARPins. Furthermore, we found that the cancer cell lines respond in a highly reproducible and defined manner to the treatment with the 441 different bivalent binding agents. The generated response profiles can thus be used for functional characterization of cell lines because they are strongly related to the cell line–specific surface receptor landscape. Thus, our method not only represents a robust tool for screening and lead identification of bispecific binding agents, but additionally offers an orthogonal approach for the functional characterization of cancer cell lines.
Data from Selective Tumor Cell Apoptosis and Tumor Regression in CDH17-Positive Colorectal Cancer Models using BI 905711, a Novel Liver-Sparing TRAILR2 Agonist
Authors: García-Martínez Juan Manuel , Wang Shirley , Weishaeupl Cordula , Wernitznig Andreas , Chetta Paolo , Pinto Catarina , Ho Jason , Dutcher Darrin , Gorman Philip N. , Kroe-Barrett Rachel , Rinnenthal Joerg , Giragossian Craig , Impagnatiello Maria Antonietta , Tirapu Iñigo , Hilberg Frank , Kraut Norbert , Pearson Mark , Kuenkele Klaus Peter
Published: April 2023

Abstract
Activation of TRAILR2 has emerged as an important therapeutic concept in cancer treatment. TRAILR2 agonistic molecules have only had limited clinical success, to date, due either to lack of efficacy or hepatotoxicity. BI 905711 is a novel tetravalent bispecific antibody targeting both TRAILR2 and CDH17 and represents a novel liver-sparing TRAILR2 agonist specifically designed to overcome the disadvantages of previous strategies. Here, we show that BI 905711 effectively triggered apoptosis in a broad panel of CDH17-positive colorectal cancer tumor cells in vitro. Efficient induction of apoptosis was dependent on the presence of CDH17, as exemplified by the greater than 1,000-fold drop in potency in CDH17-negative cells. BI 905711 demonstrated single-agent tumor regressions in CDH17-positive colorectal cancer xenografts, an effect that was further enhanced upon combination with irinotecan. Antitumor efficacy correlated with induction of caspase activation, as measured in both the tumor and plasma. Effective tumor growth inhibition was further demonstrated across a series of different colorectal cancer PDX models. BI 905711 induced apoptosis in both a cis (same cell) as well as trans (adjacent cell) fashion, translating into significant antitumor activity even in xenograft models with heterogeneous CDH17 expression. In summary, we demonstrate that BI 905711 has potent and selective antitumor activity in CDH17-positive colorectal cancer models both in vitro and in vivo. The high prevalence of over 95% CDH17-positive tumors in patients with colorectal cancer, the molecule preclinical efficacy together with its potential for a favorable safety profile, support the ongoing BI 905711 phase I trial in colorectal cancer and additional CDH17-positive cancer types (NCT04137289).
Data from Vitamin D Receptor Activation and Photodynamic Priming Enables Durable Low-dose Chemotherapy
Authors: Anbil Sriram , Pigula Michael , Huang Huang-Chiao , Mallidi Srivalleesha , Broekgaarden Mans , Baglo Yan , De Silva Pushpamali , Simeone Diane M. , Mino-Kenudson Mari , Maytin Edward V. , Rizvi Imran , Hasan Tayyaba
Published: April 2023

Abstract
Patients with cancer often confront the decision of whether to continue high-dose chemotherapy at the expense of cumulative toxicities. Reducing the dose of chemotherapy regimens while preserving efficacy is sorely needed to preserve the performance status of these vulnerable patients, yet has not been prioritized. Here, we introduce a dual pronged approach to modulate the microenvironment of desmoplastic pancreatic tumors and enable significant dose deescalation of the FDA-approved chemotherapeutic nanoliposomal irinotecan (nal-IRI) without compromising tumor control. We demonstrate that light-based photodynamic priming (PDP) coupled with vitamin D3 receptor (VDR) activation within fibroblasts increases intratumoral nal-IRI accumulation and suppresses protumorigenic CXCL12/CXCR7 crosstalk. Combined photodynamic and biochemical modulation of the tumor microenvironment enables a 75% dose reduction of nal-IRI while maintaining treatment efficacy, resulting in improved tolerability. Modifying the disease landscape to increase the susceptibility of cancer, via preferentially modulating fibroblasts, represents a promising and relatively underexplored strategy to enable dose deescalation. The approach presented here, using a combination of three clinically available therapies with nonoverlapping toxicities, can be rapidly translated with minimal modification to treatment workflow, and challenges the notion that significant improvements in chemotherapy efficacy can only be achieved at the expense of increased toxicity.