Multiple Preclinical Cancer Models Research Articles

Ataxia-Telangiectasia Mutated Loss-of-Function Displays Variant and Tissue-Specific Differences across Tumor Types.

Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

Abstract 1422: A novel gut microbiota therapeutic approach for enhancing immune checkpoint inhibitor therapy

Abstract Immune checkpoint inhibitor therapy (ICT) has revolutionized cancer care, but up to 50% of patients do not respond to ICT. A host factor implicated in variable ICT response is the gut microbiome: a) germfree/antibiotic-treated mice do not respond to ICT; b) human ICT responders have distinct gut microbiome signatures compared to non-responders; and c) fecal microbiota transplants are safe and potentially effective in cancer patients previously unresponsive to ICT. We have previously established that ICT induces translocation of gut microbiota into secondary lymphoid tissues (particularly the mesenteric lymph nodes) and tumor which is required to enhance extraintestinal anti-tumor immunity (PMID: 36867675). Here, we have developed a novel gut microbiota therapy (GMT) which circumvents the challenges and limitations of conventional microbiome therapies that use live microbes. GMT utilizes specific components derived from commensal gut microbiota identified in our published clinical study (PMID:28923537) that can be administered parenterally (subcutaneous/intravenous) to safely and reproducibly enhance ICT efficacy in multiple preclinical cancer models (B16-F10 with anti-PD-1 and/or CTLA-4, MC38 with or without anti-PD-1, humanized mouse with human melanoma and human anti-PD-1). GMT acts by agonizing the pattern recognition receptors hTLR1/2/6, hNOD2 and hSTING. GMT induces anti-tumor cytokines (IL-12) but does not significantly increase sepsis associated cytokines (IL-6, TNFa) in both human immune cells and in non-human primates. Mechanistically, GMT acts via dendritic cells, and potentially through increased expression of delta-like ligand protein 4 (DLL4), a Notch signaling ligand that activates Notch 1/4 in T cells and promotes Th1 and Th17 differentiation. Current efforts to identify the active components of GMT are underway in which we have used serial liquid-liquid extractions (ethyl acetate followed by water:methanol:chloroform) and subsequent reverse phase chromatography to identify the most active fractions and identification of the bacterial components/constituents within these fractions. Together, these results demonstrate that GMT has the potential to serve as an innate immune adjuvant (akin to vaccine adjuvants) that enhances the effect of immunotherapy for possibly many cancer types. Citation Format: Priscilla Del Valle, Gabriella Nguyen, Yongbin Choi, Parastoo Sabaeifard, Wenng li, Jake Lichterman, Laura Coughlin, Nicole Poulides, Angelica Sanjuan, Andrew Y. Koh. A novel gut microbiota therapeutic approach for enhancing immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1422.

Monopolar spindle 1 contributes to tamoxifen resistance in breast cancer through phosphorylation of estrogen receptor α.

The overexpression of mitotic kinase monopolar spindle 1 (Mps1) has been identified in many tumor types, and targeting Mps1 for tumor therapy has shown great promise in multiple preclinical cancer models. However, the role played by Mps1 in tamoxifen (TAM) resistance in breast cancer has never been reported. The sensitivity of breast cancer cells to tamoxifen was analysed in colony formation assays and wound healing assays. Enhanced transactivational activity of estrogen receptor α (ERα) led by Mps1 overexpression was determined by luciferase assays. The interaction between Mps1 and ERα was verified by co-immunoprecipitation and proximity ligation assay. Phosphorylation of ERα by Mps1 was detected by in vitro kinase assay and such phosphorylation process in vivo was proven by co-immunoprecipitation. The potential phosphorylation site(s) of ERα were analyzed by mass spectrometry. Mps1 determines the sensitivity of breast cancer cells to tamoxifen treatment. Mps1 overexpression rendered breast cancer cells more resistant to tamoxifen, while an Mps1 inhibitor or siMps1 oligos enabled cancer cells to overcome tamoxifen resistance. Mechanistically, Mps1 interacted with estrogen receptor α and stimulated its transactivational activity in a kinase activity-dependent manner. Mps1 was critical for ERα phosphorylation at Thr224 amino acid site. Importantly, Mps1 failed to enhance the transactivational activity of the ERα-T224A mutant. Mps1 contributes to tamoxifen resistance in breast cancer and is a potential therapeutic that can overcome tamoxifen resistance in breast cancer.

Abstract 1635: OKI-1546 is a selective, orally bioavailable inhibitor of CDK12 that causes tumor regression in multiple preclinical cancer models

Abstract Background: CDK12 regulates the expression of multiple genes in the DNA damage response (DDR) pathway by phosphorylating the C-terminal domain of RNA polymerase II (CTD pSer2), and it is a potential therapeutic target to exacerbate DNA damage and cell death in DDR-addicted, HER2-amplified, MYC-overexpressing, or EWS-FLI1-positive cancers. PARP inhibitor resistance is often driven by dysregulation of DDR signaling, and CDK12 inhibition may induce a state of DDR deficiency and increase the effectiveness of PARP inhibition. Materials and methods: Biochemical activity was assayed by ADP-glo (Promega), cellular proliferation was quantified by CellTiter-Glo (Promega), and CTD pSer2 was quantified by MSD immunoassay (Meso Scale Discovery). Kinase panels and in vivo studies are described in figure legends. Results: OKI-1546 has a biochemical IC50 of 15 nM against CDK12 and is highly selective for CDK12 and CDK13 when compared to other closely related CDKs; it is 649-, 81-, 11-, and 64-fold selective over CDKs 1, 2, 7, and 9, respectively. This selectivity was further confirmed via testing OKI-1546 against an affinity-based kinase panel. OKI-1546 potently inhibited proliferation of multiple cancer cell lines, with IC50 values that correlated with 50% inhibition of CTD pSer2. In CD-1 mice, OKI-1546 showed 77% oral bioavailability (%F) with a half-life of 2.7 hrs, and SD rats had 81 %F. Furthermore, a single 10 mg/kg dose reached plasma concentrations that surpassed the plasma protein binding-adjusted IC50 of CTD pSer2, which was confirmed in tumor-bearing mice showing 60% CTD pSer2 inhibition in tumors 4 hours after a single oral dose. That same dose translated into 119% tumor growth inhibition following 21 days of daily administration in the BRCA1 wild-type MDA-MB-231 tumor model and 121% tumor growth inhibition following 21 days of daily administration in the BRCA1-mutant MDA-MB-436 tumor model in mice. Four different PARP inhibitors synergistically combined with OKI-1546 to inhibit cell growth in multiple in vitro cancer models. Conclusion: OKI-1546 is a novel, covalent inhibitor of CDK12 and CDK13 that is potent, specific, orally available, and shows in vivo target engagement and tumor growth inhibition in preclinical cancer models. OKI-1546 showed activity in vivo as a single agent and cellular data suggest that inhibition of CDK12 has the potential to enhance the effects of other DDR-targeting therapeutic agents. Citation Format: Alexander A. Strait, David A. Mareska, Jyothi Mahadevan, Christie A. Schulte, Yevgeniy Izrayelit, Mark L. Boys, Richard Woessner. OKI-1546 is a selective, orally bioavailable inhibitor of CDK12 that causes tumor regression in multiple preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1635.

The Novel RXR Agonist MSU-42011 Differentially Regulates Gene Expression in Mammary Tumors of MMTV-Neu Mice

Retinoid X receptor (RXR) agonists, which activate the RXR nuclear receptor, are effective in multiple preclinical cancer models for both treatment and prevention. While RXR is the direct target of these compounds, the downstream changes in gene expression differ between compounds. RNA sequencing was used to elucidate the effects of the novel RXRα agonist MSU-42011 on the transcriptome in mammary tumors of HER2+ mouse mammary tumor virus (MMTV)-Neu mice. For comparison, mammary tumors treated with the FDA approved RXR agonist bexarotene were also analyzed. Each treatment differentially regulated cancer-relevant gene categories, including focal adhesion, extracellular matrix, and immune pathways. The most prominent genes altered by RXR agonists positively correlate with survival in breast cancer patients. While MSU-42011 and bexarotene act on many common pathways, these experiments highlight the differences in gene expression between these two RXR agonists. MSU-42011 targets immune regulatory and biosynthetic pathways, while bexarotene acts on several proteoglycan and matrix metalloproteinase pathways. Exploration of these differential effects on gene transcription may lead to an increased understanding of the complex biology behind RXR agonists and how the activities of this diverse class of compounds can be utilized to treat cancer.

Anti-tumor efficacy of a potent and selective non-covalent KRASG12D inhibitor.

Recent progress in targeting KRASG12C has provided both insight and inspiration for targeting alternative KRAS mutants. In this study, we evaluated the mechanism of action and anti-tumor efficacy of MRTX1133, a potent, selective and non-covalent KRASG12D inhibitor. MRTX1133 demonstrated a high-affinity interaction with GDP-loaded KRASG12D with KD and IC50 values of ~0.2 pM and <2 nM, respectively, and ~700-fold selectivity for binding to KRASG12D as compared to KRASWT. MRTX1133 also demonstrated potent inhibition of activated KRASG12D based on biochemical and co-crystal structural analyses. MRTX1133 inhibited ERK1/2 phosphorylation and cell viability in KRASG12D-mutant cell lines, with median IC50 values of ~5 nM, and demonstrated >1,000-fold selectivity compared to KRASWT cell lines. MRTX1133 exhibited dose-dependent inhibition of KRAS-mediated signal transduction and marked tumor regression (≥30%) in a subset of KRASG12D-mutant cell-line-derived and patient-derived xenograft models, including eight of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models. Pharmacological and CRISPR-based screens demonstrated that co-targeting KRASG12D with putative feedback or bypass pathways, including EGFR or PI3Kα, led to enhanced anti-tumor activity. Together, these data indicate the feasibility of selectively targeting KRAS mutants with non-covalent, high-affinity small molecules and illustrate the therapeutic susceptibility and broad dependence of KRASG12D mutation-positive tumors on mutant KRAS for tumor cell growth and survival.

CUB Domain-Containing Protein 1 (CDCP1) is a rational target for the development of imaging tracers and antibody-drug conjugates for cancer detection and therapy.

Rationale: An antibody-drug conjugate (ADC) is a targeted therapy consisting of a cytotoxic payload that is linked to an antibody which targets a protein enriched on malignant cells. Multiple ADCs are currently used clinically as anti-cancer agents significantly improving patient survival. Herein, we evaluated the rationale of targeting the cell surface oncoreceptor CUB domain-containing protein 1 (CDCP1) using ADCs and assessed the efficacy of CDCP1-directed ADCs against a range of malignant tumors.Methods: CDCP1 mRNA expression was evaluated using large transcriptomic datasets of normal/tumor samples for 23 types of cancer and 15 other normal organs, and CDCP1 protein expression was examined in 34 normal tissues, >300 samples from six types of cancer, and in 49 cancer cell lines. A recombinant human/mouse chimeric anti-CDCP1 antibody (ch10D7) was labelled with 89Zirconium or monomethyl auristatin E (MMAE) and tested in multiple pre-clinical cancer models including 36 cancer cell lines and three mouse xenograft models.Results: Analysis of CDCP1 expression indicates elevated CDCP1 expression in the majority of the cancers and restricted expression in normal human tissues. Antibody ch10D7 demonstrates a high affinity and specificity for CDCP1 inducing cell signalling via Src accompanied by rapid internalization of ch10D7/CDCP1 complexes in cancer cells. 89Zirconium-labelled ch10D7 accumulates in CDCP1 expressing cells enabling detection of pancreatic cancer xenografts in mice by PET imaging. Cytotoxicity of MMAE-labelled ch10D7 against kidney, colorectal, lung, ovarian, pancreatic and prostate cancer cells in vitro, correlates with the level of CDCP1 on the plasma membrane. ch10D7-MMAE displays robust anti-tumor effects against mouse xenograft models of pancreatic, colorectal and ovarian cancer.Conclusion: CDCP1 directed imaging agents will be useful for selecting cancer patients for personalized treatment with cytotoxin-loaded CDCP1 targeting agents including antibody-drug conjugates.

Abstract 1747: Induction of nucleosome disassembly with small molecules as a novel anti-cancer approach

Abstract CBL0137 is a clinical-stage anti-cancer drug candidate that is active against multiple preclinical cancer models. It is a carbazole-based DNA intercalator, discovered by cell-based screening for p53 activation in cells lacking DNA damage. Binding of curaxin to DNA results in alterations in DNA length, charge, and flexibility, which together impair the ability of DNA to wrap around the histone core. Treatment of cells with CBL0137 leads to decondensation of chromatin accompanied by histone eviction. This causes the binding of histone chaperone FACT to epitopes normally hidden within nucleosomes and the activation of p53 by FACT-bound CK2. p53 activation occurs in the absence of detectable DNA damage. However, CBL0137 is also toxic to p53-deficient cells, suggesting that its toxicity is mediated by events upstream of p53 activation. Trying to understand the mechanisms of CBL0137 toxicity, we found that FACT is essential for the viability of tumor but not normal cells. FACT was discovered as a transcription elongation factor performing disassembly and reassembly of nucleosomes in cell-free conditions. However, no reduction of transcription is observed with FACT depletion in cells. Moreover, measurement of FACT chromatin binding and gene expression upon FACT loss suggest that FACT negatively regulates gene expression. mammalian FACT does not disassemble nucleosomes during transcription but binds “open” nucleosomes, with partially unwrapped DNA, appearing during transcription, and tethers nucleosome components together to prevent histone loss from chromatin. In cells treated with CBL0137 nucleosomes are mostly lost in regions enriched with tandem AC/TG dinucleotide repeats, known as microsatellites. These regions accumulate FACT in treated cells. To understand the effects of FACT loss from other chromatin regions, we measured nucleosome distribution in cells with genetically-depleted FACT. Regions normally enriched with FACT lose nucleosomes in a transcription-dependent manner. Thus, curaxin induces chromatin disassembly via a direct effect on nucleosomal DNA and inhibition of FACT-mediated nucleosome restoration at transcribed regions. To explain the reasons for the higher toxicity of curaxin in tumor versus normal cells, we hypothesized that chromatin is, in general, less stable in tumor than in normal cells. Thus, less drug is needed to cause unrepairable nucleosome disassembly in tumor cells. We compared chromatin organization in syngeneic normal, immortalized, and transformed cells and found that during tumor progression, there is loss of histones, reduction of condensed chromatin and increase of DNA accessibility to the transcriptional machinery. The same signs are observed in human tumors. In summary, curaxin kills cells via the destabilization of nucleosomes and disassembly of chromatin, a phenomenon that we have named “chromatin damage.” Tumor cells are more sensitive to chromatin damage due to their preexisting, less stable chromatin. Citation Format: Alfiya Safina, Poorva Sandlesh, Jianmin Wang, Katerina V. Gurova. Induction of nucleosome disassembly with small molecules as a novel anti-cancer approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1747.

Abstract 3620: Tumor targeted delivery of glutamine antagonist: Use of CES1-/- mice

Abstract 6-diazo-5-oxo-L-norleucine (DON), a potent glutamine antagonist, broadly blocks glutamine utilizing reactions critical for the synthesis of nucleic acids, amino acids, proteins and the generation of alpha-ketoglutarate for energy metabolism. DON has shown robust efficacy in multiple preclinical cancer models and exploratory clinical studies. Although promising, development of DON was halted due to its dose-limiting gastrointestinal (GI)-toxicities, as the GI system is highly dependent on glutamine utilization. Given DON’s promising efficacy, we developed novel tumor cell-targeted glutamine antagonists intended to circulate intact as inert prodrug/s in plasma and be preferentially biotransformed to DON in tumor cells. Our prodrug strategy of utilizing elevated protease activities in cancer tissues (e.g. Histone deacetylase, Cathepsin-L) led to the discovery of a highly innovative prodrug pharmacophore. Using a well-defined screening paradigm, we discovered compound 6, (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido) hexanamido)-6-diazo-5-oxohexanoate), that showed stability in plasma, liver and intestinal homogenates, yet was readily cleaved to DON in P493B human tumor cells. When directly compared to DON, compound 6, exhibited a 55-fold enhanced P493B cell-to-plasma ratio. In a time-dependent study, compound 6 showed sustained DON delivery to P493B cells while maintaining minimal release in human plasma. Moreover, in a cell proliferation assay, compound 6 showed dose-dependent inhibition of P493B cell growth. One challenging aspect of prodrug development is the selection of appropriate animal model, as it can be confounded by interspecies variation in metabolism, specifically in the varying levels of the carboxylesterase enzyme, CES1, which is highly abundant in rodent plasma but not present in human plasma. We hypothesized that CES1-/- mice would recapitulate human metabolism and serve as a suitable model for our prodrug containing ester promoiety on its carboxylate. Using plasma from CES1-/- mice, wild-type mice and human, we confirmed that compound 6 exhibited similar stability in CES1-/- mice and human plasma but not in wild-type mice plasma. We then performed pharmacokinetic evaluation in C57BL/6 CES1-/- mice bearing flank murine EL4 tumors. Following subcutaneous dosing (1mg/kg DON equivalent), compound 6 exhibited excellent pharmacokinetics with a ~5-fold higher DON tumor exposures (AUC= 5.1 nmol/g*h) versus plasma (1.1 nmol/ml*h) and a 11-fold higher tumor exposures versus GI-tissues (toxicity site; AUC = 0.45 nmol/ml*h). These studies describe discovery of a tumor targeted glutamine antagonist. In addition, we introduce a murine model, that recapitulates human metabolism and can be broadly utilized in prodrug development. Future studies will investigate the dose dependent efficacy and safety of compound 6 in tumor bearing C57BL/6 CES1-/- mice. Citation Format: Rana Rais, Jesse Alt, Ranjeet Dash, Lukáš Tenora, Pavel Majer, Barbara S. Slusher. Tumor targeted delivery of glutamine antagonist: Use of CES1-/- mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3620.

Abstract A21: Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27 dependent pathway

Abstract Purpose: Agonist antibodies targeting the T cell co-stimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across multiple preclinical models of cancer. In the clinic, however, development of these agents has been stymied by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist driven anti-tumor immunity from hepatotoxicity. Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immune competent mice, with or without co-administration of checkpoint blockade, via measurement of serum transaminase levels, through imaging of liver immune infiltrates, and via qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines and chemokines. Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27, which is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage and their removal dramatically exacerbates 4-1BB agonist hepatitis. Co-administration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma. Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation of myeloid cells to produce Interleukin-27. Co-administration of CTLA-4 and/or CCR2 blockade may minimize hepatitis but yields equal or greater antitumor immunity. Citation Format: Ashvin R. Jaiswal, Todd Bartkowiak, Casey R. Ager, Renee Chin, Chao Hsien Chen, Pratha Budhani, Midan Ai, Matthew J. Reilley, Manu M. Sebastian, David Hong, Michael A. Curran. Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27 dependent pathway [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A21.

Infiltrating T Cells Increase IDO1 Expression in Glioblastoma and Contribute to Decreased Patient Survival.

Purpose: Indoleamine 2,3 dioxygenase 1 (IDO1) mediates potent immunosuppression in multiple preclinical models of cancer. However, the basis for elevated IDO1 expression in human cancer, including the most common primary malignant brain tumor in adults, glioblastoma (GBM), is poorly understood. The major objective of this study is to address this gap in our understanding of how IDO1 expression contributes to the biology of GBM, and whether its level of expression is a determinant of GBM patient outcome.Experimental Design: Patient-resected GBM, The Cancer Genome Atlas, human T-cell:GBM cocultures, as well as nu/nu, NOD-scid, and humanized (NSG-SGM3-BLT) mice-engrafted human GBM form the basis of our investigation.Results:In situ hybridization for IDO1 revealed transcript expression throughout patient-resected GBM, whereas immunohistochemical IDO1 positivity was highly variable. Multivariate statistical analysis revealed that higher levels of IDO1 transcript predict a poor patient prognosis (P = 0.0076). GBM IDO1 mRNA levels positively correlated with increased gene expression for markers of cytolytic and regulatory T cells, in addition to decreased patient survival. Humanized mice intracranially engrafted human GBM revealed an IFNγ-associated T-cell-mediated increase of intratumoral IDO1Conclusions: Our data demonstrate that high intratumoral IDO1 mRNA levels correlate with a poor GBM patient prognosis. It also confirms the positive correlation between increased GBM IDO1 levels and human-infiltrating T cells. Collectively, this study suggests that future efforts aimed at increasing T-cell-mediated effects against GBM should consider combinatorial approaches that coinhibit potential T-cell-mediated IDO1 enhancement during therapy. Clin Cancer Res; 23(21); 6650-60. ©2017 AACR.

A Novel Tumor-Activated Prodrug Strategy Targeting Ferrous Iron Is Effective in Multiple Preclinical Cancer Models.

Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics.

85 - RXDX-105 demonstrates anti-tumor efficacy in multiple preclinical cancer models driven by molecular alterations in RET or BRAF oncogenes

85 - RXDX-105 demonstrates anti-tumor efficacy in multiple preclinical cancer models driven by molecular alterations in RET or BRAF oncogenes

All-Atom Molecular Dynamics Reveals Mechanism of Zinc Complexation with Therapeutic F10.

Advancing the use of therapeutic nucleic acids requires understanding the chemical and structural properties that allow these polymers to promote the death of malignant cells. Here we explore Zn2+ complexation by the fluoropyrimidine polymer F10, which has strong activities in multiple preclinical models of cancer. Delivery of fluoropyrimidine FdUMP in the 10-residue polymer F10 rather than the nucleobase (5-fluorouracil) allows consideration of metal ion binding effects on drug delivery. The differences in metal ion interactions with fluoropyrimidine compared to normal DNA results in conformation changes that affect protein binding, cell uptake, and codelivery of metals such as zinc, and the cytoxicity thereof. Microsecond-time-scale, all-atom simulations of F10 predict that zinc selectively stabilizes the polymer via interactions with backbone phosphate groups and suggest a mechanism of complexation for the zinc-base interactions shown in previous experimental work. The positive zinc ions are attracted to the negatively charged phosphate groups. Once the Zn2+ ions are near F10, they cause the base's N3 nitrogen to deprotonate. Subsequently, magnesium atoms displace zinc from their interactions with phosphate, freeing the zinc ions to interact with the FdU bases by forming weak interactions with the O4 oxygen and the fluorine attached to C5. These interactions of magnesium with phosphate groups and zinc with nucleobases agree with previous experimental results and are seen in MD simulations only when magnesium is introduced after N3 deprotonation, indicating a specific order of metal binding events. Additionally, we predict interactions between zinc and F10's O2 atoms, which were not previously observed. By comparison to 10mers of polyU and polydT, we also predict that the presence of fluorine increases the binding affinity of zinc to F10 relative to analogous strands of RNA and DNA consisting of only native nucleotides.

TLR and NLRP3 inflammasome-dependent innate immune responses to tumor-derived autophagosomes (DRibbles).

Autophagosomes derived from tumor cells, also referred to as defective ribosomal products in blebs (DRibbles), have been previously shown to stimulate potent T-cell responses and mediate tumor regression when used as therapeutic cancer vaccines in multiple preclinical cancer models. In this report, we investigated the underlining mechanisms by which DRibbles induced T-cell activation, particularly how DRibbles activated antigen-presenting cells (APCs). We found that DRibbles could induce a rapid differentiation of monocytes and DC precursor (pre-DC) cells into functional APCs. DRibbles triggered innate receptor signaling via Toll-like Receptors (TLR)-2, TLR4, TLR7, TLR8, and nucleotide-binding oligomerization domain-containing protein 2 (NOD2), but not TLR3, TLR5, or TLR9. DRibbles induced PBMCs to produce pro-inflammatory cytokines, such as IL-6, IL-10, TNF-α, and IL-1β. DRibbles induced IL-1β release from PBMC or THP-1 cells without LPS priming, but required the core machinery of NLRP3 inflammasomes. Active endocytosis was required for inflammasome activation and cross presentation, and blocking endosome acidification or the ER-associated degradation (ERAD) pathway resulted in opposite effects on these two processes. Our data show that DRibbles could induce strong innate immune responses via multiple pattern recognition receptors, and explain why DRibbles could function as excellent antigen carriers to induce adaptive immune responses to both tumor cells and viruses. In contrast to the well-established inhibitory effect of autophagy on the inflammasome activation of APCs, our study demonstrates that isolated autophagosomes (DRibbles) from antigen donor cells activate inflammasomes by providing first and second signals required for IL-1β production by PMBC.

Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models.

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.

Abstract 2468: Antibody targeting soluble NKG2D ligand sMIC induces regression of primary tumors and eliminates metastasis in multiple pre-clinical cancer models

Abstract Expression of the MHC I-chain related molecules A and B (MICA/B) on epithelial cell surface in response to transformation or DNA damage can signal the immune system of the abnormality and thus initiate active immune surveillance by Natural Killer (NK) cells and cytotoxicity T cells. We and others have shown that malignant tumor cells can shed MICA/B to down regulate NKG2D expression and negatively impact NK and CD8 T cell function in cancer patients. Increased tumor-specific shedding of cell surface NKG2D ligand, MHC I chain related molecule (MIC), is associated with advanced stage and metastasis in many types of epithelial cancer. High serum levels of soluble MIC (sMIC) insults the immune system not only by down-regulating NKG2D expression on natural killer (NK) cells and effector T cells but also perturbing NK cell peripheral maintenance. Whether sMIC is an effective cancer therapeutic target has not been addressed due to the lack of specific sMIC-blocking reagent and clinically relevant pre-clinical animal models. Using a “humanized” clinically relevant spontaneous prostate carcinoma TRAMP/MIC bi-transgenic mouse model and multiple engineered syngeneic transplantable tumor models, we unprecedentedly show that therapy with a sMIC-specific monoclonal antibody induced rapid regression of primary tumors and metastasis without systemic toxicity. The therapy revamped a myriad of anti-tumor immune responses including NK cell homeostatic renewal and function, CD4 T cells to Th1 and Th17 responses. Using the well-defined melanoma model, we further show that anti-sMIC therapy augments Pmel-1 antigen-specific CD8 T cell responses. Notably, depletion of NK cells mitigated the therapeutic effect of anti-sMIC antibody and the effector function of both CD4 and CD8 T cells. We conclude that sMIC is an effective therapeutic target to potentiate innate and adoptive immune responses against MIC+ malignancies. Citation Format: Jennifer D. Wu, Fahmin Basher, Mark Rubinstein. Antibody targeting soluble NKG2D ligand sMIC induces regression of primary tumors and eliminates metastasis in multiple pre-clinical cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2468. doi:10.1158/1538-7445.AM2015-2468

Profiling Hsp90 differential expression and the molecular effects of the Hsp90 inhibitor IPI-504 in high-grade glioma models.

Retaspimycin hydrochloride (IPI-504), an Hsp90 (heat shock protein 90) inhibitor, has shown activity in multiple preclinical cancer models, such as lung, breast and ovarian cancers. However, its biological effects in gliomas and normal brain derived cellular populations remain unknown. In this study, we profiled the expression pattern of Hsp90α/β mRNA in stable glioma cell lines, multiple glioma-derived primary cultures and human neural stem/progenitor cells. The effects of IPI-504 on cell proliferation, apoptosis, motility and expression of Hsp90 client proteins were evaluated in glioma cell lines. In vivo activity of IPI-504 was investigated in subcutaneous glioma xenografts. Our results showed Hsp90α and Hsp90β expression levels to be patient-specific, higher in high-grade glioma-derived primary cells than in low-grade glioma-derived primary cells, and strongly correlated with CD133 expression and differentiation status of cells. Hsp90 inhibition by IPI-504 induced apoptosis, blocked migration and invasion, and significantly decreased epidermal growth factor receptor levels, mitogen-activated protein kinase and/or Akt activities, and secretion of vascular endothelial growth factor in glioma cell lines. In vivo study showed that IPI-504 could mildly attenuate tumor growth in immunocompromised mice. These findings suggest that targeting Hsp90 by IPI-504 has the potential to become an active therapeutic strategy in gliomas in a selective group of patients, but further research into combination therapies is still needed.

Abstract P2-16-22: A dose-finding phase lb study of BEZ235 in combination with paclitaxel in patients with HER2-negative, locally advanced or metastatic breast cancer

Abstract Background: The PI3K/Akt/mTOR pathway is the most frequently activated signaling pathway in breast cancer (BC). Activation of this pathway promotes tumor growth and progression, and resistance to anticancer therapies, including paclitaxel (PTX). BEZ235 is an oral, ATP-competitive inhibitor that targets class l PI3K and downstream effectors mTORC1/2. BEZ235 has shown antiproliferative, proapoptotic, and antiangiogenic activity in multiple preclinical cancer models. In a Ph I study, single-agent BEZ235 given twice daily (BID) showed preliminary signs of clinical activity. Methods: This was a multicenter, open-label Ph Ib/II study of daily, oral BEZ235 given BID in combination with weekly (QW) intravenous PTX in women with HER2-, metastatic or locally advanced BC (NCT01495247). For the Ph Ib part, the primary objective was to determine the maximum tolerated dose (MTD)/recommended Ph II dose (RP2D) of BEZ235 in combination with PTX based on dose-limiting toxicities (DLTs) using a 5-parameter adaptive Bayesian logistic regression model with overdose control. MTD was defined as the highest dose not causing medically unacceptable DLTs in &amp;gt;35% of pts during Cycle 1 (28 days). Secondary objectives included safety, preliminary activity, and PK. Safety was monitored by physical exams, vital signs, laboratory tests, weight, and cardiac health. AEs were assessed continuously by CTCAE v4.03. Tumor evaluations were performed at screening and every 8 wks after starting treatment, and assessed locally by RECIST v1.1. Results: As of April 12, 2013, 18 pts (mean age: 50 yr; 67% ≥4 prior lines of antineoplastic therapy) had been enrolled into Ph Ib. 13 pts received BEZ235 200 mg BID + PTX 80 mg/m2 QW. The dose level was then reduced to BEZ235 100 mg BID + PTX 80 mg/m2 QW (n = 5). Of 17 evaluable pts (12 at the 200-mg BID dose; 5 at the 100-mg BID dose), 6 (35%) developed ≥1 DLTs (5 [42%] at the 200-mg BID dose; 1 [20%] at the 100-mg BID dose): neutropenia (n = 3 [1 at the 100-mg BID dose]), stomatitis (n = 3), nausea (n = 2), and vomiting (n = 1). Of 18 pts, 12 have discontinued (6 due to disease progression; 4 AEs; 1 physician decision; 1 pt/guardian decision). Median duration of exposure to BEZ235 and PTX was 76 days each. The most common suspected study drug-related AEs included asthenia (78%), nausea (72%), neutropenia (72%), diarrhea (67%), stomatitis (61%), and decreased appetite (56%). Among 13 pts receiving BEZ235 200 mg BID, 2 (15%) achieved a PR, 4 (31%) had SD, and 4 (31%) had PD; all 5 pts receiving BEZ235 100 mg BID had SD. PK results showed large interpatient variability at both dose levels. Analysis of pAKT and p4EBP1 was performed on peripheral blood mononuclear cells collected from pts before treatment and 4 h following treatment (Cmax). PK/PD analysis did not provide evidence of a strong and consistent correlation between BEZ235 plasma concentrations and pAKT inhibition. Conclusions: The MTD/RP2D of BEZ235 in combination with PTX in pts with locally advanced or metastatic HER2- BC was not reached. Based on the observed overall safety profile, efficacy, variable PK, and PD data from the Ph Ib part of this study, further enrollment has stopped. The Ph II part of the study will not be conducted. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-16-22.

Abstract LB-317: Potent in vivo pharmacology of AZD9150, a next-generation, constrained ethyl-modified antisense oligonucleotide targeting STAT3 in multiple preclinical cancer models.

Abstract Next generation sequencing technologies have greatly expanded our understanding of cancer genomes, epigenomes and transcriptomes. This knowledge, however, has not yet been effectively translated into improved cancer therapeutics, partly due to the inability of available therapeutic modalities to target the most promising cancer driver pathways. In contrast to other therapeutic approaches, the druggable universe is not limited with antisense technology as inhibitors can be rationally designed based on sequence information alone. Recent human clinical data has demonstrated potent activity of systemically-administered, unformulated, antisense oligonucleotides (ASOs) when targeted to liver expressed genes. However, robust activity in extra-heptatic tissues and tumors has been limited with existing ASO chemistries. Here we evaluate the activity of high affinity next generation (constrained ethyl) ASOs in extrahepatic tissues and tumors of multiple preclinical cancer models including spontaneous tumors, human tumor xenografts and several primary patient-derived xenograft models. As a test case we employed next generation ASOs to inhibit the difficult to drug transcription factor STAT3. ASOs targeting mouse STAT3 sequences and the human-specific STAT3 ASO (AZD9150) demonstrate potent and selective inhibition of target RNA and protein levels in tumors and tumor-associated stromal cells of a broad range of cancer models, resulting in strong antitumor activity in several models. These findings suggest that next generation ASO technology is now poised to become a key therapeutic modality to bridge the pharmacogenomic divide in cancer drug discovery. The STAT3 ASO inhibitor, AZD9150 is currently in human clinical studies including patients with lymphomas. Citation Format: Youngsoo Kim, Jeff Hsu, Tianyuan Zhou, Nancy Zhang, Richard Woessner, Murali VP Nadella, Deborah Lawson, Corinne Reimer, Guobin He, Joanna Schmidt, Xiaokun Xiao, Sarah Greenlee, Gourab Bhattacharjee, Gene Hung, Brett P. Monia, A. Robert MacLeod. Potent in vivo pharmacology of AZD9150, a next-generation, constrained ethyl-modified antisense oligonucleotide targeting STAT3 in multiple preclinical cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-317. doi:10.1158/1538-7445.AM2013-LB-317