Human Xenograft Mouse Model Research Articles

Abstract A053: Inhibition of the MEK5-ERK5 signaling pathway blocks pancreatic tumorigenesis

Abstract Mutant KRAS represents one of the most common driving events in pancreatic ductal adenocarcinomas (PDAC), yet therapies targeting this pathway have proven largely ineffective. We identified ERK5 activation as critical component of KRAS-driven pancreatic tumorigenesis. ERK5 belongs to the MAP kinase family and is phosphorylated and activated by the MAPK kinase MEK5. Therefore, the aim of this study is to elucidate the function of the MEK5-ERK5 signaling axis in PDAC pathogenesis. Several novel genetically engineered mouse models with pancreas-specific deletion or activation of the MEK5-ERK5 signaling were generated to study pancreatic cancer initiation and progression. Caerulein-induced pancreatitis was performed in p48Cre/+ KrasG12D/+ (Kras) and p48Cre/+ KrasG12D/+ Erk5fl/fl (Kras;Erk5) mice to study early steps in pancreatic tumorigenesis. To investigate if ERK5 ablation impacts tumor progression a two-stage tumor model utilizing dual recombinase (Cre/Flp) mouse model was utilized. Next, the impact of ERK5 depletion or pharmacological inhibition on survival of pancreatic cancer mice was analyzed. Furthermore, human patient-derived PDAC xenograft models in immunodeficient mice were utilized to evaluate efficacy of a small molecule ERK5 inhibitor which does not exhibit off-target activity on bromodomains. Deletion of ERK5 or inhibition of its kinase activity resulted in blocked tumorigenesis in Kras mutant mice using caerulein-induced pancreatitis. Upon pancreatitis induction pancreas in Kras;Erk5 mice exhibit normal tissue architecture with significantly fewer proliferating cells compared to control mice. Intriguingly, pancreas-specific expression of constitutively active MEK5 (MEK5DD) even without oncogenic Kras mutations was sufficient in promoting development of precancerous lesions after pancreatitis induction. Next, we observed that genetic depletion or pharmacological inhibition of ERK5 significantly prolonged survival of the KPC PDAC mouse model, whereas expression of MEK5DD in KPC mice resulted in significantly accelerated tumorigenesis and reduced survival compared to control mice. Furthermore, depletion of ERK5 in already established PDAC resulted in significantly prolonged survival and reduced tumor burden indicating an important role of ERK5 in tumor maintenance. Finally, ERK5 inhibitor treatment of patient-derived PDAC xenografts in immunocompromised mice resulted in tumor growth arrest. Our studies demonstrate that the MEK5-ERK5 signaling pathway plays an important role in pancreatic cancer initiation and progression. Deletion of ERK5 or pharmacological inhibition of ERK5 kinase activity results in significantly reduced tumor burden and prolonged survival in autochthonous PDAC models and patient-derived xenografts. Conversely, activation of MEK5-ERK5 signaling is sufficient to drive pancreatic tumorigenesis indicating an essential role of the ERK5 pathway as downstream effector of oncogenic KRAS. Further studies will be performed to identify the mechanisms of ERK5 action including downstream kinase targets. Citation Format: Simone Hausmann, Asli Ece Kasapoglu, Ana Benitez Morales, Shane Lofgren, Pawel K. Mazur. Inhibition of the MEK5-ERK5 signaling pathway blocks pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A053.

MYCN Regulates Cell Survival Via EIF4G1 in Inv(16) Acute Myeloid Leukemia

Transcription factors are frequently deregulated in leukemia and are pivotal to its pathogenesis. The core-binding factor (CBF) transcription factor is a heterodimer of CBFβ and RUNX proteins, is critical to hematopoiesis, and dysregulated in AML. The fusion oncogene CBFβ-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells by sequestering RUNX1 by protein-protein interaction to induce AML. The CBFβ-SMMHC inhibitor, AI-10-49, specifically disrupts its binding to RUNX1 resulting in genome-wide RUNX1 binding, apoptosis induction, and enhanced survival in inv(16) mouse model [Illendula* and Pulikkan* et al, Science 2015]. While previous studies have demonstrated that AI-10-49 induced cell death is partly mediated by repression of MYC expression in inv(16) AML cells [Pulikkan et al, Cell 2018], the function of other MYC family members, in inv(16) cell survival, and overall in AML, remains poorly understood. By conducting Real Time RT-PCR in CD34+ bone marrow cells from human AML samples, we identified MYCN upregulation in inv(16) AML. Analysis of RNA-seq data from inv(16) AML cells treated with AI-10-49 revealed that MYCN expression is downregulated (7-fold). MYCN silencing via CRISPR/Cas9 induced apoptosis in inv(16) AML cells. Furthermore, MYCN genetic targeting by CRISPR/Cas9-RNP approach in human primary inv(16) AML cells transplanted in humanized NSGS xenograft mouse model displayed significant delay in leukemia development, underlining the role of MYCN in inv(16) AML maintenance. ChIP-seq analysis conducted in inv(16) AML cells treated with AI-10-49 demonstrated increased RUNX1 occupancy at MYCN distal genomic element located at +26 Kb (named 'MYCN-e26') downstream from MYCN TSS without any RUNX1 binding at MYCN TSS. ChIP-seq analysis for histone marks such as H3K27ac and H3K4Me1, and chromatin accessibility by ATAC-seq suggested MYCN-e26 acts as a transcriptionally active enhancer element. Analysis of DNase I hypersensitive sites (DHSs) representing accessible chromatin regions in primary AML samples revealed that MYCN-e26 is a bona fide AML enhancer. Finally, deletion of a genomic element in MYCN-e26 surrounding the RUNX1 binding site (200 bp) by CRISPR/Cas9 genome editing downregulated MYCN transcript levels and inhibited inv(16) AML cell survival. Taken together, MYCN-e26 is instrumental in MYCN transcription and cell survival in inv(16) AML cells as a RUNX1 target. To identify the mechanism(s) by which MYCN regulates inv(16) AML cell survival, we performed CUT&Tag sequencing for MYCN and H3K27ac histone mark in inv(16) AML cells. Gene ontology analysis for MYCN binding peaks in transcriptionally active regions revealed mRNA metabolism and stability as the major pathways orchestrated by MYCN in inv(16) AML cells. By comparing the mRNA metabolism/ stability signature genes with transcripts significantly deregulated in human primary inv(16) AML samples, we identified eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) as one of the major genes regulated by MYCN in inv(16) AML cell survival. Our CUT&Tag sequencing analysis demonstrated MYCN binds to EIF4G1 promoter and enhances its transcription. MYCN silencing by CRISPR/Cas9 approach in inv(16) AML cells showed significant downregulation of EIF4G1, suggesting MYCN is required for EIF4G1 expression in inv(16) AML cells. EIF4G1 genetic silencing via CRISPR/Cas9 approach induced apoptosis in inv(16) AML cells in vitro, and significantly delayed leukemia development in NSGS xenograft mouse model, suggesting EIF4G1 is required for the survival of inv(16) leukemia-initiating cells. Our studies demonstrate that CBFβ-SMMHC modulates MYCN levels by inhibition of RUNX1 binding to MYCN distal enhancer in inv(16) AML cells. In addition, our studies bring novel function of MYCN in inv(16) leukemic cell survival by regulating EIF4G1. Future therapeutic approaches targeting MYCN/EIF4G1 axis in combination with AI-10-49 warrant better clinical outcome for the treatment of inv(16) AML.

CD84: A Novel Target for CAR T-Cell Therapy for Acute Myeloid Leukemia

Relapsed or refractory acute myeloid leukemia (AML) patients have scarce treatment options despite the recent emergence of novel targeted therapies. Chimeric antigen receptor (CAR) T-cell therapies have been approved for the treatment of B-cell malignancies and multiple myeloma, but it remains very challenging to treat myeloid malignancies with this therapeutic modality, in part due to the absence of specific tumor antigens in these indications. Antigens expressed on AML cells are usually shared with healthy hematopoietic stem/progenitor cells (HSPC) and targeting them can lead to life-threatening on-target/off-tumor toxicity. CD84 antigen is an immunoreceptor member of the SLAM family (SLAMF5), whose expression has been reported in certain immune cells and in B-cell malignancies. In this study, we show for the first time that CD84 is overexpressed in AML and present the first results of CD84-targeting CAR T-cells for the treatment of this disease. We analyzed AML primary samples and found that CD84 is overexpressed on the surface of AML cells, both de novo and at relapse, while lower expression was detected in CD34+ HSPC. Furthermore, immunohistochemistry analysis of healthy human tissues showed that CD84 is not expressed in the parenchyma of liver, kidney, lung, myocardium, skin, or brain (cerebral cortex and cerebellum), although expression was detected in tissue monocytes/macrophages. This expression pattern is similar to that of CD123, a clinical stage and safe CAR T target in AML. Anti-CD84 CAR T cells were engineered using both fully human and murine CD84-binding sequences. The sequences of the variable regions of the light (VL) and the heavy (VH) chain were used to design different single-chain variable fragments (scFv), changing the order (VLVH or vice versa) and the linker length. In total, we designed 21 lentiviral second-generation CARs with an anti-CD84 scFv, a CD8α hinge and transmembrane domain, a 4-1BB costimulatory and a CD3ζ signaling domain. Some constructs were discarded due to failed lentiviral transduction or target recognition; the following CAR T-cells were characterized in depth: one with a fully human scFv, CD84.01 (VHVL), and 5 with different murine scFv, CD84.02 (VLVH), CD84.03 (VHVL), CD84.04 (VHVL), CD84.05 (VHVL), and CD84.06 (VHVL). All these CAR T-cells displayed cytotoxic activity towards, and were able to proliferate and secrete cytokines in vitro when exposed to the CD84high Kasumi-1 and the CD84low U937 AML cell lines (Figure 1A). We then analyzed their cytotoxicity against monocytes, B cells and T cells purified from the same donor as the CD84 CAR T cells were generated; all CART cells exerted some cytotoxic activity against their own T cells, which was much lower than that against Kasumi-1 cells. To assess the myeloablative potential of CD84 CAR T cells, we carried out cytotoxic assays against CD34+ HSPCs purified from 1) donor cord blood; 2) directly from donor bone marrow samples, or 3) an apheresis product of a donor for an allogeneic hematopoietic stem cell transplant. We found that the cytotoxic effect against CD34+ HSPC was much lower than that against leukemic cells. Based on these in vitro results, the CARTs CD84.02 and CD84.03 were tested for their efficacy in a human NSG xenograft mouse model of AML (Kasumi-1). Although CD84.02 appeared to be more effective than CD84.03, both were able to control the disease for at least 43 days (Figure 1B), at which point no leukemic cells were detected in the bone marrow and spleen of CAR T-treated animals. In contrast, CD84 CAR T cells were found in these organs at the end of the experiment. In summary, here we report that CART cells directed against CD84 can eliminate AML cells both in vitro and in vivo. Our data support the therapeutic use of CD84 CAR T cells for relapsed/refractory (R/R) AML. However, due to its potential myelotoxicity, we encourage its first use in a clinical trial as a bridge therapy to allogeneic stem cell transplant for patients with R/R AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Role of Integrin Beta-1 in the Chemoresistance of Acute Myeloid Leukemia

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive disease that has a poor prognosis with a survival rate of 30-40%. The bone marrow microenvironment provides protection to AML cells from chemotherapy and thus confers cell adhesion-mediated drug resistance (CAM-DR) and promotes relapse. Integrins are key anchor molecules in CAM-DR of leukemia cells; they are heterodimeric adhesion molecules on the cell surface consisting of an alpha and a beta unit. Upregulated integrin alpha 4 and 6 expression is associated with poor survival in leukemias. We hypothesize that the targeting of integrin beta 1 (ITGB1, β1, or CD29) will also affect the integrin alpha units as they are heterodimer partners. This could be a superior approach to overcome CAM-DR in AML than just targeting the individual alpha units. METHODS: ITGB1 gene was knocked out via CRISPR-Cas9 using 2 guide RNAs in AML cell lines and expression of integrin subunits was profiled by flow cytometry. AML cell lines were treated in vitro with the anti-human ITGB1 monoclonal antibody OS2966 in combination with Ara-C chemotherapy and we determined effects on cell viability by flow cytometry and on cell signaling using RNA-sequencing. Using human patient-derived AML xenograft mouse models, we tested if blockade of ITGB1 combined with chemotherapy can overcome CAM-DR in vivo. Mice were treated with control total Ig (10 mg/kg), OS2966 (10 mg/kg), total Ig + Ara-C (600 mg/kg), or OS2966 + Ara-C twice a week for two weeks. RESULTS: Analysis of patient-derived AML cells at diagnosis and relapse, and of AML cell lines, showed high expression of ITGB1 and varying levels of other alpha and beta units. Loss-of-function studies of ITGB1 using CRISPR/Cas9-mediated knockout of ITGB1 in MLL-rearranged and FLT3-ITD+ AML cell lines MV4-11 and MOLM-13, and in AML cell line U937, revealed that surface expression of integrin alphas 3, 4, 5 and 6 were markedly decreased as determined by flow cytometry analysis. ITGB2 expression was high in all 3 AML cell lines and not affected by ITGB1 deletion. Remarkably, ITGB3 was downregulated upon ITGB1 deletion in 2 out of 3 AML cell lines, whereas the 3rd AML cell line did not express any ITGB3. As a result of the ITGB1 deletion, AML cells showed less adhesion to murine bone marrow stroma (OP9) cells and to extracellular matrix proteins. We found that inhibition of surface ITGB1 on human AML using OS2966 de-adhered the AML cells from OP9 cells. Moreover, ITGB1 blockade using OS2966 synergized with Ara-C in vitro and significantly decreased cell viability compared to single agent controls in human AML cell lines U937 and MV4-11. RNA sequencing data suggest that the NFkB pathway is significantly dysregulated in the combination treatment Ara-C and OS2966 compared to Ara-C alone. To test ITGB1 inhibition in vivo, a patient-derived AML sample, HEM279 (Fig. 1A), and AML cell line, U937 (Fig. 1B), were engrafted into NSG-SGM3 mice. Leukemic mice were treated with single agents including control total Ig, OS2966, or Ara-C; and with total Ig in combination with Ara-C or OS2966. In both xenograft models, the combination treatment of OS2966 with Ara-C significantly prolonged survival of leukemic mice compared to Ara-C only: HEM279: MST 115 +/- 15.4 days vs 73.1 +/- 5.2 days post-AML injection; p=0.0005; U937: MST 170 days vs 32 days post-AML injection; p=0.0004; 50% of the mice engrafted with U937 and treated with OS2966 with Ara-C survived until day 290 post-AML injection, which is when the experiment was terminated. CONCLUSIONS: Integrin alpha subunit expression is dependent on ITGB1 expression, supporting our hypothesis that targeting integrin beta 1 affects alpha integrin units. ITGB1 inhibition with OS2966 combined with chemotherapy significantly decreases cell viability of AML cell lines in vitro and prolongs survival of xenograft mice in vivo. Our findings will allow for better understanding of the role of ITGB1 in drug resistance of AML and of ITGB1 as a preclinical target in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DHODH inhibition impedes glioma stem cell proliferation, induces DNA damage, and prolongs survival in orthotopic glioblastoma xenografts.

Glioma stem cells (GSCs) promote tumor progression and therapeutic resistance and exhibit remarkable bioenergetic and metabolic plasticity, a phenomenon that has been linked to their ability to escape standard and targeted therapies. However, specific mechanisms that promote therapeutic resistance have been somewhat elusive. We hypothesized that because GSCs proliferate continuously, they may require the salvage and de novo nucleotide synthesis pathways to satisfy their bioenergetic needs. Here, we demonstrate that GSCs lacking EGFR (or EGFRvIII) amplification are exquisitely sensitive to de novo pyrimidine synthesis perturbations, while GSCs that amplify EGFR are utterly resistant. Furthermore, we show that EGFRvIII promotes BAY2402234 resistance in otherwise BAY2402234 responsive GSCs. Remarkably, a novel, orally bioavailable, blood-brain-barrier penetrating, dihydroorotate dehydrogenase (DHODH) inhibitor BAY2402234 was found to abrogate GSC proliferation, block cell-cycle progression, and induce DNA damage and apoptosis. When dosed daily by oral gavage, BAY2402234 significantly impaired the growth of two different intracranial human glioblastoma xenograft models in mice. Given this observed efficacy and the previously established safety profiles in preclinical animal models and human clinical trials, the clinical testing of BAY2402234 in patients with primary glioblastoma that lacks EGFR amplification is warranted.

Theaflavin-3,3 ′ -Digallate Plays a ROS-Mediated Dual Role in Ferroptosis and Apoptosis via the MAPK Pathway in Human Osteosarcoma Cell Lines and Xenografts

Globally, osteosarcoma (OS) is the most prevalent form of primary bone cancer in children and adolescents. Traditional neoadjuvant chemotherapy regimens have reached a bottleneck; thus, OS survivors have unsatisfactory outcomes. Theaflavin-3,3'-digallate (TF3) exhibits potent anticancer properties against many human cancers. Nevertheless, the biological effects and the underlying molecular mechanism of TF3 in human OS remain unclear. The objective of this study was to investigate the effects of TF3 on human OS cell lines and mouse xenograft models. The results showed that TF3 reduced cell viability, suppressed cell proliferation, and caused G0/G1 cell cycle arrest in both MG63 and HOS cell lines in a concentration-dependent manner. TF3 also altered the homeostatic mechanisms for iron storage in the examined cell lines, resulting in an excess of labile iron. Unsurprisingly, TF3 caused oxidative stress through reduced glutathione (GSH) exhaustion, reactive oxygen species (ROS) accumulation, and the Fenton reaction, which triggered ferroptosis and apoptosis in the cells. TF3 also induced MAPK signalling pathways, including the ERK, JNK, and p38 MAPK pathways. Furthermore, oxidative stress was shown to be the primary reason for TF3-induced proliferation inhibition, programmed cell death, and MAPK pathway activation in vitro. Moreover, TF3 exhibited markedly strong antitumour efficacy in vivo in mouse models. In summary, this study demonstrates that TF3 concomitantly plays dual roles in apoptotic and ferroptotic cell death by triggering the ROS and MAPK signalling pathways in both in vitro and in vivo models.

Pharmacokinetics and Pharmacodynamics of TAK-164 Antibody Drug Conjugate Coadministered with Unconjugated Antibody.

The development of new antibody-drug conjugates (ADCs) has led to the approval of 7 ADCs by the FDA in 4years. Given the impact of intratumoral distribution on efficacy of these therapeutics, coadministration of unconjugated antibody with ADC has been shown to improve distribution and efficacy of several ADCs in high and moderately expressed tumor target systems by increasing tissue penetration. However, the benefit of coadministration in low expression systems is less clear. TAK-164, an ADC composed of an anti-GCC antibody (5F9) conjugated to a DGN549 payload, has demonstrated heterogeneous distribution and bystander killing. Here, we evaluated the impact of 5F9 coadministration on distribution and efficacy of TAK-164 in a primary human tumor xenograft mouse model. Coadministration was found to improve the distribution of TAK-164 within the tumor, but it had no significant impact (increase or decrease) on efficacy. Experimental and computational evidence indicates that this was not a result of tumor saturation, increased binding to perivascular cells, or compensatory bystander effects. Rather, the cellular potency of DGN549 was matched with the single-cell uptake of TAK-164 making its IC50 close to its equilibrium binding affinity (KD), and as such, coadministration dilutes total DGN549 in cells below the maximum cytotoxic concentration, thereby offsetting an increased number of targeted cells with decreased ability to kill each cell. These results provide new insights on matching payload potency to ADC delivery to help identify when increasing tumor penetration is beneficial for improving ADC efficacy and demonstrate how mechanistic simulations can be leveraged to design clinically effective ADCs.

Whole transcriptome profiling of liquid biopsies from tumour xenografted mouse models enables specific monitoring of tumour-derived extracellular RNA.

While cell-free DNA (cfDNA) is widely being investigated, free circulating RNA (extracellular RNA, exRNA) has the potential to improve cancer therapy response monitoring and detection due to its dynamic nature. However, it remains unclear in which blood subcompartment tumour-derived exRNAs primarily reside. We developed a host-xenograft deconvolution framework, exRNAxeno, with mapping strategies to either a combined human-mouse reference genome or both species genomes in parallel, applicable to exRNA sequencing data from liquid biopsies of human xenograft mouse models. The tool enables to distinguish (human) tumoural RNA from (murine) host RNA, to specifically analyse tumour-derived exRNA. We applied the combined pipeline to total exRNA sequencing data from 95 blood-derived liquid biopsy samples from 30 mice, xenografted with 11 different tumours. Tumoural exRNA concentrations are not determined by plasma platelet levels, while host exRNA concentrations increase with platelet content. Furthermore, a large variability in exRNA abundance and transcript content across individual mice is observed. The tumoural gene detectability in plasma is largely correlated with the RNA expression levels in the tumour tissue or cell line. These findings unravel new aspects of tumour-derived exRNA biology in xenograft models and open new avenues to further investigate the role of exRNA in cancer.

Mechanism of Cordyceps militaris against non-small cell lung cancer: based on serum metabolomics

This study investigated the potential mechanism of Cordyceps militaris(CM) against non-small cell lung cancer(NSCLC) based on serum untargeted metabolomics. Specifically, Balb/c nude mice were used to generate the human lung cancer A549 xenograft mouse model. The tumor volume, tumor weight, and tumor inhibition rate in mice in the model, cisplatin, Cordyceps(low-, medium-, and high-dose), and CM(low-, medium-, and high-dose) groups were compared to evaluate the influence of CM on lung cancer. Gas chromatography-mass spectrometry(GC-MS) was used for the analysis of mouse serum, SIMCA 13.0 for the compa-rison of metabolic profiles, and MetaboAnalyst 5.0 for the analysis of metabolic pathways. According to the pharmacodynamic data, the tumor volume and tumor weight of mice in high-dose CM group and cisplatin group decreased as compared with those in the model group(P<0.05 or P<0.01). The results of serum metabolomics showed that the metabolic profiles of the model group were significantly different from those of the high-dose CM group, and the content of endogenous metabolites was adjusted to different degrees. A total of 42 differential metabolites and 7 differential metabolic pathways were identified. In conclusion, CM could significantly inhibit the tumor growth of lung cancer xenograft mice. The mechanism is the likelihood that it influences the aminoacyl-tRNA biosynthesis, the metabolism of D-glutamine and D-glutamate, metabolism of alanine, aspartate, and glutamate, metabolism of glyoxylate and dicarboxylic acid, biosynthesis of phenylalanine, tyrosine, and tryptophan, arginine biosynthesis as well as nitrogen metabolism. This study elucidated the underlying mechanism of CM against NSCLC from the point of metabolites. The results would lay a foundation for the anticancer research and clinical application of CM.

High CAR intensity of expression confers enhanced antitumor effect against lymphoma without functional exhaustion.

Identifying factors that ameliorates clinical outcomes following CART therapy represents an unmet need. We hypothesized that CAR expression level would have a significant impact on CART efficacy and tested this with CAR30+ TSCM-LIKE enriched cells. By sorting T-cells according to CAR mean fluorescence intensity in two markedly different populations (CARHI and CARLO), we showed that a high CAR expression enhances antitumor efficacy in vitro, that is sustained after sequential re-exposures to tumor cells and is not associated with T-cell exhaustion or differentiation. Furthermore, we found a correlation between high surface CAR expression and antitumor effect with CAR19+ T-cells, thus validating our findings with CAR30. Definitive proof of CARHI T-cells improved antitumor efficacy was demonstrated in a human Hodgkin's lymphoma xenograft mouse model, where CAR30-TSCM-LIKE enriched products with high intensity of CAR expression achieved superior tumor control in vivo and longer survival than those with a low intensity of CAR expression. Our data suggest that modulation of CAR intensity of expression represents an additional strategy to increase CART therapy clinical efficacy.

Discovery of a 2,6-diarylpyridine-based hydroxamic acid derivative as novel histone deacetylase 8 and tubulin dual inhibitor for the treatment of neuroblastoma

Herein, two series of HDAC/tubulin dual inhibitors via introducing the key pharmacophore of HDAC inhibitor into the skeletons of 2,6-diarylpyridine and 2′-arylchalcone were synthesized. Among them, 2,6-diarylpyridine-based hydroxamic acid 10a exhibited good inhibitory activity against HDAC8 (IC50 = 117 nM) with 50-fold and 42-fold high selectivity relative to HDAC1 and HDAC6, respectively. Meanwhile, 10a disrupted tubulin polymerization effectively and exhibited potent antiproliferative activity against BE-(2)-C cell line, with IC50 value of 17 nM. Mechanism studies revealed that 10a blocked cell cycle, induced cellular apoptosis and suppressed colony formation. Moreover, 10a possessed good physicochemical properties and metabolic stability. Importantly, 10a exhibited better antitumor effects in human neuroblastoma xenograft mice model than those of clinical HDAC inhibitor and tubulin inhibitor, whether used alone or in combination. These results highlighted the advantages of the HDAC8/tubulin dual inhibitor 10a as an outstanding antitumor agent.

Docetaxel enhances the therapeutic efficacy of PSMA-specific CAR-T cells against prostate cancer models by suppressing MDSCs.

Prostate cancer can undergo curative effects by radical prostatectomy or radical radiotherapy. However, the best treatment for more aggressive high-risk prostate cancer remains controversial. Insufficient infiltration capacity and dysfunction are commonly occurrences in engineered T lymphocytes expressing chimeric antigen receptor (CAR-T), characterizing cancer immunotherapy failure. We conducted this study to investigate whether the combinative application of docetaxel and PSMA-CAR-T cells could be a more effective treatment to prostate cancer. Expressions of prostate specific membrane antigen (PSMA) on prostate cancer cells were examined by Flow cytometry. The efficaciousness of PSMA-CAR-T was evaluated in vitro using ELISA and RTCA. The effect of intermixed therapy was assessed in vivo utilizing a human prostate cancer liver metastasis mouse model and a human prostate cancer cell xenograft mouse model. The outcome of cytokine discharge and cell killing assays demonstrated that PSMA-CAR-T cells have characteristic effector capacity against PSMA+ prostate cancer cells in vitro. Additionally, collaborative treatment of PSMA-CAR-T cells and docetaxel have cooperative efficacy in a mouse model of human prostate cancer. The merged strategy could be seen as an undeveloped avenue to augmenting adoptive CAR-T cell immunotherapy and mitigating the adverse side effects of chemotherapy. Cooperation of PSMA-specific CAR-T cells and the chemotherapy drug docetaxel can impressively ameliorate antitumor effectiveness against an installed metastatic human prostate cancer model in NPG mice.

Robbing Peter to Pay Paul: Chlorhexidine gluconate demonstrates short-term efficacy and long-term cytotoxicity.

Wound cleansing agents are routine in wound care and preoperative preparation. Antiseptic activity intends to prevent contaminating microbes from establishing an infection while also raising concerns of cytotoxicity and delayed wound healing. We evaluated the cytotoxicity of five clinically used wound cleaning agents (saline, povidone iodine, Dove® and Dial® soaps, and chlorhexidine gluconate [CHG]) using both an ex vivo and in vivo human skin xenograft mouse model, in contrast to classical in vitro models that lack the structural and compositional heterogeneity of human skin. We further established an ex vivo wound contamination model inoculated with ~100 cells of Pseudomonas aeruginosa or Staphylococcus aureus to evaluate antimicrobial efficacy. Scanning electron microscopy and confocal microscopy were used to evaluate phenotypic and spatial characteristics of bacterial cells in wound tissue. CHG significantly reduced metabolic activity of the skin explants, while all treatments except saline affected local cellular viability. CHG cytotoxicity persisted and progressed over 14 days, impairing wound healing in vivo. Within the contamination model, CHG treatment resulted in a significant reduction of P. aeruginosa wound surface counts at 24 h post‐treatment. However, this effect was transient and serial application of CHG had no effect on both P. aeruginosa or S. aureus microbial growth. Microscopy revealed that viable cells of P. aeruginosa reside deep within wound tissue post‐CHG application, likely serving as a reservoir to re‐populate the tissue to a high bioburden. We reveal concerning cytotoxicity and limited antimicrobial activity of CHG in human skin using clinically relevant models, with the ability to resolve spatial localization and temporal dynamics of tissue viability and microbial growth.

DNA sequence-selective G-A cross-linking ADC payloads for use in solid tumour therapies

Antibody-Drug Conjugates (ADCs) are growing in importance for the treatment of both solid and haematological malignancies. There is a demand for new payloads with novel mechanisms of action that may offer enhanced therapeutic efficacy, especially in patients who develop resistance. We report here a class of Cyclopropabenzindole-Pyridinobenzodiazepine (CBI-PDD) DNA cross-linking payloads that simultaneously alkylate guanine (G) and adenine (A) bases in the DNA minor groove with a defined sequence selectivity. The lead payload, FGX8-46 (6), produces sequence-selective G-A cross-links and affords cytotoxicity in the low picomolar region across a panel of 11 human tumour cell lines. When conjugated to the antibody cetuximab at an average Drug-Antibody Ratio (DAR) of 2, an ADC is produced with significant antitumour activity at 1 mg/kg in a target-relevant human tumour xenograft mouse model with an unexpectedly high tolerability (i.e., no weight loss observed at doses as high as 45 mg/kg i.v., single dose).

Farrerol exhibits inhibitory effects on lung adenocarcinoma cells by activating the mitochondrial apoptotic pathway.

Farrerol is an herbal compound extracted from rhododendron. Here, our study is to investigate biological effects of farrerol on lung adenocarcinoma (LAC) cells. Human LAC cell lines and xenograft mouse model were utilized to define the effects of farrerol on tumor growth. Our findings indicated that farrerol significantly reduced LAC cell viability as well as the colony-forming capacity. Flow cytometry analysis demonstrated that farrerol contributed to cell apoptosis and G0/G1 phase cell cycle arrest. Mechanistically, farrerol treatment upregulated proapoptotic molecules (Bak, Bid, cleaved caspase-3 and cleaved caspase-9) and senescence markers (p16 and p2), but downregulated antiapoptosis genes (Bcl-2 and Bcl-XL) and cell cycle-associated genes (CyclinD1 and CDK4); meanwhile, the phosphorylation of retinoblastoma (Rb) protein was attenuated upon pretreatment of LAC cells with farrerol in comparison to untreated control. Further studies indicated that farrerol elevated reactive oxygen species levels, activating mitochondrial apoptotic pathway and causing cell apoptosis. However, exposure to farrerol did not result in significant apoptosis in normal lung epithelial cells, suggesting a tumor-specific effect of farrerol on LAC cells. In animal model, farrerol showed a significant inhibitory effect on LAC xenograft tumor growth. And gene expressions in tumor tissues, as mentioned above, were in line with the in vitro results. Taken together, these results suggested that farrerol caused LAC cell apoptosis by activating mitochondrial apoptotic pathway, whereas farrerol treatment had no notable effect on normal lung epithelial cells. Farrerol might be an effective therapeutic drug for LAC.

Combination of [177Lu]Lu-DOTA-TATE Targeted Radionuclide Therapy and Photothermal Therapy as a Promising Approach for Cancer Treatment: In Vivo Studies in a Human Xenograft Mouse Model.

Peptide receptor radionuclide therapy (PRRT) relies on α- and β-emitting radionuclides bound to a peptide that commonly targets somatostatin receptors (SSTRs) for the localized killing of tumors through ionizing radiation. A Lutetium-177 (177Lu)-based probe linked to the somatostatin analog octreotate ([177Lu]Lu-DOTA-TATE) is approved for the treatment of certain SSTR-expressing tumors and has been shown to improve survival. However, a limiting factor of PRRT is the potential toxicity derived from the high doses needed to kill the tumor. This could be circumvented by combining PRRT with other treatments for an enhanced anti-tumor effect. Photothermal therapy (PTT) relies on nanoparticle-induced hyperthermia for cancer treatment and could be a useful add-on to PRRT. Here, we investigate a strategy combining [177Lu]Lu-DOTA-TATE PRRT and nanoshell (NS)-based PTT for the treatment of SSTR-expressing small-cell lung tumors in mice. Our results showed that the combination treatment improved survival compared to PRRT alone, but only when PTT was performed one day after [177Lu]Lu-DOTA-TATE injection (one of the timepoints examined), showcasing the effect of treatment timing in relation to outcome. Furthermore, the combination treatment was well-tolerated in the mice. This indicates that strategies involving NS-based PTT as an add-on to PRRT could be promising and should be investigated further.

Abstract 2902: Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies

Abstract CD20 and CD37 are co-expressed on most B-NHL and CLL tumors. Systemic depletion of normal and malignant B-cells by anti-CD20 antibodies (e.g. rituximab and obinutuzumab) and anti-CD37 antibodies (e.g. otlertuzumab and BI 836826) has been well tolerated in clinical studies. Depletion of B-cells is reversible, with the ability to repopulate B-cells from hematopoietic stem cells. Dual targeting CD20 and CD37 may improve the clinical efficacy and decrease drug resistance. PSB202 is a novel biological entity composed of two engineered monoclonal antibodies which are produced from a single stable CHO cell line - a humanized anti-CD20 antibody (PSB102) and a humanized anti-CD37 antibody (PSB107) at a 1:1 molecular ratio. In vitro binding studies demonstrated that PSB202 specifically binds to human CD20 and CD37. The antibodies’ binding to FcγRIIIa was enhanced by reducing the fucose level on each antibody component. PSB202 directly induced potent autonomous killing via target engagement. PSB202 mediated depletion of human and monkey blood B-cells and malignant human B-cell lines through ADCC activity at EC50 of double digit pM and CDC activity at EC50 of low single digit nM. Importantly, PSB202 effectively depleted B-cells in ex vivo cultures of blood cells from CLL and NHL patients who were refractive to rituximab treatment. The antitumor activity of PSB202 was demonstrated in human xenograft models in mice. PSB202 was able to reduce different human lymphoma tumors by up to ~97.9% in a dose-dependent manner, suggesting that PSB202 might be potentially efficacious against multiple types of B-cell malignancies. Synergy was demonstrated for PSB202 in combination with lenalidomide. Toxicokinetic assessment in cynomolgus monkeys showed a dose proportional exposure for both antibody components, with mean terminal elimination half-life (T1/2) of ~60 and ~68 hours for the anti-CD20 and anti-CD37 component, respectively. PBS202 was well tolerated, with observed toxicities mostly related to its activity as a B-cell depletion agent. As expected, PSB202 when administered at ≥ 10 mg/kg resulted in nearly complete depletion of B lymphocytes 24 hours following the 1st dose. B-cells returned to near baseline within 2 months after the last dose at 10 mg/kg. PSB202 can deplete normal and malignant B-cells by targeting two clinically validated targets through multiple distinct mechanisms including direct B-cell killing, enhanced ADCC activity, and CDC activity. PSB202 offers the potential to improve clinical efficacy, decrease drug resistance, provide administration convenience especially when combining with additional therapeutics, lower the cost for treatment, and reduce the unwanted side effects from chemotherapy. PSB202 is currently being evaluated in patients with previously treated, relapsed, indolent B-cell malignancies (NCT05003141). Citation Format: Paul Algate, Zhi Liu, Saying Yuan, Hua Liu, Cristina Domeier, Haiming Jiang, Yuanzhi Lao, Yanling Xu, William C. Fanslow, Ron Schoner, Wei Yan. Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell 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 2902.

Abstract 5458: Pharmacodynamics of WX-0593 combined with bevacizumab biosimilar on human lung cancer xenograft mouse models

Abstract Objective: To evaluate the antitumor activity of WX-0593 (anaplastic lymphoma kinase/ROS proto-oncogene 1 receptor tyrosine kinase [ALK/ROS1] tyrosine kinase inhibitor) combined with bevacizumab biosimilar in vivo. Methods: Cell line-derived xenograft (CDX) model: NCI-H3122 cell suspension was subcutaneously inoculated into the right axilla of BALB/c-nu mice. When the average tumor volume reached 100-300 mm3, the mice were randomly divided into six groups (eight mice/group) and administered with solvent, WX-0593 1.5 or 5 mg/kg, bevacizumab biosimilar 1 mg/kg, or WX-0593 1.5 or 5 mg/kg + bevacizumab biosimilar 1 mg/kg, respectively. Patient-derived xenografts (PDX) model: LU-01-1443 tumor tissue blocks of 20-30 mm3 were subcutaneously inoculated into the right axilla of BALB/c-nu mice. When the average tumor volume reached 115 mm3, the mice were randomly divided into four groups (eight mice/group) and administered with solvent, WX-0593 2.5 mg/kg, bevacizumab biosimilar 1.5 mg/kg or their combination. Tumor diameter was measured twice a week. The tolerability was also assessed. Results: CDX tumor model: There were significant differences between each group and the control group (all P <0.01). The combination of WX-0593 5 mg/kg and bevacizumab biosimilar 1 mg/kg was significantly more effective than WX-0593 5 mg/kg alone or bevacizumab biosimilar 1 mg/kg alone (both P <0.01). PDX tumor model: Compared with the control group, single-agent WX-0593 2.5 mg/kg and bevacizumab biosimilar 1.5 mg/kg delayed tumor growth. The tumor suppressive effect of WX-0593 combined with bevacizumab biosimilar was impressive (treated/control ratio =11.81%; tumor growth inhibition =92.19%) and significantly better than WX-0593 2.5 mg/kg or bevacizumab biosimilar 1.5 mg/kg alone (P =0.004, 0.003, respectively). Toxicity: There were no abnormalities in appearance and mental state in each group during the experiment, and the weight of the mice remained stable. Conclusions: The combination of WX-0593 and bevacizumab biosimilar had obvious synergistic effect and showed potent tumor suppressive activity, which was significantly better than WX-0593 or bevacizumab biosimilar alone at the same dose. There was no drug-related toxicity in all groups, and no increase in toxicity after combined administration. Citation Format: Shansong Zheng, Qingmei Zheng, Xin Dong, Xinmei Wang, Dongmei Chen, Huicheng Duan, Cuicui Ma, Chenwei Zhang. Pharmacodynamics of WX-0593 combined with bevacizumab biosimilar on human lung cancer xenograft mouse 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 5458.

Abstract 160: IKKα interacts with DARPP-32 to promote lung adenocarcinoma growth

Abstract Lung cancer is the leading cause of cancer-related death in the U.S. and worldwide. Non-small cell lung cancer (NSCLC) represents 80-85% of all lung cancer diagnoses, most of which present as advanced disease with poor prognoses. Despite existing treatment options, the overall 5-year survival rate of lung cancer patients is about 17%. Many patients are diagnosed with advanced stage disease that frequently progresses after developing resistance to therapy. Therefore, a better understanding of the molecular mechanisms that control NSCLC progression is necessary to develop new therapies to improve the prognoses of patients. We recently demonstrated that dopamine and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated splice variant, t-DARPP, promote NSCLC growth using functional studies, orthotopic mouse models, and patient-derived specimens. Specifically, we previously reported that DARPP-32 isoforms promote NSCLC cell migration through activation of non-canonical NF-κB2 signaling by establishing a direct physical interaction with inhibitory kappa B kinase-alpha (IKKα). Therefore, we first sought to determine whether IKKα directly phosphorylates DARPP-32. Indeed, we observed that kinase active IKKα protein phosphorylates DARPP-32 protein at the Thr-34 position using in vitro kinase assays. Given that phosphorylated DARPP-32 acts as a potent inhibitor of protein phosphatase 1 (PP1), we assessed PP1 activity upon transient overexpression of IKKα in DARPP-32-overexpressed NSCLC cells. Our findings suggest phosphorylation of DARPP-32 by IKKα results in decreased PP1-mediated phosphatase activity. This DARPP-32-mediated inhibition of PP1 was not observed in NSCLC cells stably overexpressing a form of DARPP-32 that cannot phosphorylated at the Thr-34 residue (via T34A mutation), suggesting IKKα protein phosphorylates DARPP-32 at the Thr-34 position to stimulate DARPP-32-mediated inhibition of PP1 activity. Correspondingly, we have observed increased phosphorylation of ERK protein, a kinase regulated by PP1, upon pharmacological inhibition of PP1 as well as following overexpression of IKKα protein. Ablation of IKKα via lentiviral transduction reduces NSCLC cell growth in soft-agar colony formation assays. Furthermore, using an orthotopic human NSCLC cell line-derived xenograft mouse model, we have shown that depletion of IKKα reduces tumor growth. Taken together, our findings suggest that IKKα contributes to NSCLC growth by interacting with DARPP-32 to stimulate oncogenic kinase activity through the inhibition of PP1 phosphatase activity. Citation Format: Sk. Kayum Alam, Li Wang, Zhu Zhu, Luke H. Hoeppner. IKKα interacts with DARPP-32 to promote lung adenocarcinoma growth [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 160.

Abstract 3104: Inverse correlation of circulating CD8+ T cells with tumor burden from immune checkpoint treatment in humanized orthotopic model of renal cell carcinoma

Abstract Background: Renal cell carcinoma (RCC) is a highly heterogeneous and metastatic disease with a widely varying prognosis. RCC is the first solid tumor treated with immune checkpoint blockade and approved by the FDA for showing improvement of clinical outcomes. Immune checkpoints physiologically regulate immune response via appropriate signals to T cells and maintain homeostasis, while pathologically they can be co-opted by cancer cells to evade the immune system. Here we developed a humanized (Hu)-mice model with adaptive transferred human immune cells to test the role of ICB [anti-PD-1/PD-L1, or anti-CTLA-4 antibody (Ab)] in an orthotopic human RCC tumor-bearing xenograft mouse model and analyzed the correlation of innate immune cells with tumor burden. Methods: Luciferase-tagged RCC cell line 786-O were injected intra-kidney into immune-deficient Rag2 mice. After tumor formation, mice were humanized by intraperitoneal injection of donor PBMCs (20×106/per mouse). Treatment groups included control, anti-PD-1/PD-L1 Abs or anti-CTLA-4 Ab (all Abs 200 µg/mouse, iv) weekly for 4 weeks. Tumor growth was measured by weekly bioluminescent imaging (BLI) and tumor weight at necropsy. The presence of human immune cells and tumor infiltrating lymphocytes was confirmed by flow cytometry and immunohistochemistry staining. The significance of the differences between groups was determined by student t test. The Pearson correlation coefficient was obtained by analysis of correlation between different study parameters. Results: BLI level showed a steady xenograft tumor progression for nine weeks. Anti-PD-1/PD-L1 Abs treatment trended toward reducing tumor BLI level while anti-CTLA-4 Ab significantly decreased tumor BLI level (p<0.01). Furthermore, anti-PD-1/PD-L1 Abs reduced lung metastasis with a significant decrease of BLI level compared to control group (p<0.05). Meanwhile, humanization was evidenced by robust, elevated levels of human CD45+ lymphocytes in the peripheral blood compared to non-reconstituted Rag2 mice. Infiltrated human CD3+CD8+ cells were found in mouse blood and spleen. Granzyme B+ cells, CD31+ cells, and pan-cytokeratin+ cells were found in tumors. Interestingly, we observed inverse correlation between circulating human CD8+ T cells and mouse RCC tumor burden. Conclusion: We successfully generated a humanized RCC mouse model which allows the RCC xenograft to grow steadily within the human immune system reconstituted Rag2 mice. Circulating CD8+ T cells are inversely correlated to tumor growth in this model. The PD-1/PD-L1 or CTLA-4-targeted immunotherapies showed distinct therapeutic effects in our humanized orthotopic xenograft model, which may lead to personalized treatment targeting different immune checkpoints to reinvigorate anti-tumor responses in RCC patients. Citation Format: Xin Zhang, Amita Bhattarai, Ravan Moret, Grace Maresh, Alicia Nicole Ray, David Woods, Rachel Graham, Maria Latis, Stephen Bardot, Li Li. Inverse correlation of circulating CD8+ T cells with tumor burden from immune checkpoint treatment in humanized orthotopic model of renal cell carcinoma [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 3104.