Brk/PTK6 and Involucrin Expression May Predict Breast Cancer Cell Responses to Vitamin D3.

The process of human embryonic mammary development gives rise to the structures in which mammary cells share a developmental lineage with skin epithelial cells such as keratinocytes. As some breast carcinomas have previously been shown to express high levels of involucrin, a marker of keratinocyte differentiation, we hypothesised that some breast tumours may de-differentiate to a keratinocyte-derived 'evolutionary history'. To confirm our hypothesis, we investigated the frequency of involucrin expression along with that of Brk, a tyrosine kinase expressed in up to 86% of breast carcinomas whose normal expression patterns are restricted to differentiating epithelial cells, most notably those in the skin (keratinocytes) and the gastrointestinal tract. We found that involucrin, a keratinocyte differentiation marker, was expressed in a high proportion (78%) of breast carcinoma samples and cell lines. Interestingly, tumour samples found to express high levels of involucrin were also shown to express Brk. 1,25-dihydroxyvitamin D3, a known differentiation agent and potential anti-cancer agent, decreased proliferation in the breast cancer cell lines that expressed both involucrin and Brk, whereas the Brk/involucrin negative cell lines tested were less susceptible. In addition, responses to 1,25-dihydroxyvitamin D3 were not correlated with vitamin D receptor expression. These data contribute to the growing body of evidence suggesting that cellular responses to 1,25-dihydroxyvitamin D3 are potentially independent of vitamin D receptor status and provide an insight into potential markers, such as Brk and/or involucrin that could predict therapeutic responses to 1,25-dihydroxyvitamin D3.

HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.

HMGB1 mediates invasion and PD-L1 expression through RAGE-PI3K/AKT signaling pathway in MDA-MB-231 breast cancer cells.

BACKGROUND: High-mobility group box 1 (HMGB1) is increased in breast cancer cells as the result of exposure to the secreted substances from cancer-associated fibroblasts and plays a crucial role in cancer progression and drug resistance. Its effect, however, on the expression of programmed death ligand 1 (PD-L1) in breast cancer cells has not been investigated. This study aimed to investigate the mechanism of HMGB1 through receptors for advanced glycation end products (RAGE) on cell migration/invasion and PD-L1 expression in breast cancer cells. METHODS: A 3-dimensional (3-D) migration and invasion assay and Western blotting analysis to evaluate the function and the mechanism under recombinant HMGB1 (rHMGB1) treatment with knockdown of RAGE using shRAGE and PI3K/AKT inhibitors was performed. RESULTS: The results revealed that rHMGB1 induced MDA-MB-231 cell migration and invasion. The knockdown of RAGE using shRAGE and PI3K/AKT inhibitors attenuated 3-D migration and invasion in response to rHMGB1 compared to mock cells. PD-L1 up-regulation was observed in both parental MDA-MB-231 (P) and MDA-MB-231 metastasis to bone marrow (BM) cells treated with rHMGB1, and these effects were alleviated in RAGE-knock down (KD) breast cancer cells as well as in PI3K/AKT inhibitor-treated cells. CONCLUSIONS: Collectively, these findings indicate that HMGB1-RAGE through PI3K/AKT signaling promotes not only breast cancer cell invasion but also PD-L1 expression which leads to the destruction of the effector T cells. The attenuating HMGB1-RAGE-PI3K/AKT pathway may help to attenuate breast cancer cell aggressive phenotypes.

Breast Tumour Kinase (Brk/PTK6) Contributes to Breast Tumour Xenograft Growth and Modulates Chemotherapeutic Responses In Vitro.

Breast tumour kinase (Brk/PTK6) is overexpressed in up to 86% of breast cancers and is associated with poorer patient outcomes. It is considered a potential therapeutic target in breast cancer, even though the full spectrum of its kinase activity is not known. This study investigated the role of the kinase domain in promoting tumour growth and its potential in sensitising triple negative breast cancer cells to standard of care chemotherapy. Triple negative human xenograft models revealed that both kinase-inactive and wild-type Brk promoted xenograft growth. Suppression of Brk activity in cells subsequently co-treated with the chemotherapy agents doxorubicin or paclitaxel resulted in an increased cell sensitivity to these agents. In triple negative breast cancer cell lines, the inhibition of Brk kinase activity augmented the effects of doxorubicin or paclitaxel. High expression of the alternatively spliced isoform, ALT-PTK6, resulted in improved patient outcomes. Our study is the first to show a role for kinase-inactive Brk in human breast tumour xenograft growth; therefore, it is unlikely that kinase inhibition of Brk, in isolation, would halt tumour growth in vivo. Breast cancer cell responses to chemotherapy in vitro were kinase-dependent, indicating that treatment with kinase inhibitors could be a fruitful avenue for combinatorial treatment. Of particular prognostic value is the ratio of ALT-PTK6:Brk expression in predicating patient outcomes.

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.

Metabolomic changes of the multi (-AGC-) kinase inhibitor AT13148 in cells, mice and patients are associated with NOS regulation.

INTRODUCTION: To generate biomarkers of target engagement or predictive response for multi-target drugs is challenging. One such compound is the multi-AGC kinase inhibitor AT13148. Metabolic signatures of selective signal transduction inhibitors identified in preclinical models have previously been confirmed in early clinical studies. This study explores whether metabolic signatures could be used as biomarkers for the multi-AGC kinase inhibitor AT13148. OBJECTIVES: To identify metabolomic changes of biomarkers of multi-AGC kinase inhibitor AT13148 in cells, xenograft / mouse models and in patients in a Phase I clinical study. METHODS: HILIC LC-MS/MS methods and Biocrates AbsoluteIDQ™ p180 kit were used for targeted metabolomics; followed by multivariate data analysis in SIMCA and statistical analysis in Graphpad. Metaboanalyst and String were used for network analysis. RESULTS: BT474 and PC3 cells treated with AT13148 affected metabolites which are in a gene protein metabolite network associated with Nitric oxide synthases (NOS). In mice bearing the human tumour xenografts BT474 and PC3, AT13148 treatment did not produce a common robust tumour specific metabolite change. However, AT13148 treatment of non-tumour bearing mice revealed 45 metabolites that were different from non-treated mice. These changes were also observed in patients at doses where biomarker modulation was observed. Further network analysis of these metabolites indicated enrichment for genes associated with the NOS pathway. The impact of AT13148 on the metabolite changes and the involvement of NOS-AT13148- Asymmetric dimethylarginine (ADMA) interaction were consistent with hypotension observed in patients in higher dose cohorts (160-300 mg). CONCLUSION: AT13148 affects metabolites associated with NOS in cells, mice and patients which is consistent with the clinical dose-limiting hypotension.

Quizartinib-resistant FLT3-ITD acute myeloid leukemia cells are sensitive to the FLT3-Aurora kinase inhibitor CCT241736.

Internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML); it causes constitutive activation of FLT3 kinase and is associated with high relapse rates and poor survival. Small-molecule inhibition of FLT3 represents an attractive therapeutic strategy for this subtype of AML, although resistance from secondary FLT3 tyrosine kinase domain (FLT3-TKD) mutations is an emerging clinical problem. CCT241736 is an orally bioavailable, selective, and potent dual inhibitor of FLT3 and Aurora kinases. FLT3-ITD+ cells with secondary FLT3-TKD mutations have high in vitro relative resistance to the FLT3 inhibitors quizartinib and sorafenib, but not to CCT241736. The mechanism of action of CCT241736 results in significant in vivo efficacy, with inhibition of tumor growth observed in efficacy studies in FLT3-ITD and FLT3-ITD-TKD human tumor xenograft models. The efficacy of CCT241736 was also confirmed in primary samples from AML patients, including those with quizartinib-resistant disease, which induces apoptosis through inhibition of both FLT3 and Aurora kinases. The unique combination of CCT241736 properties based on robust potency, dual selectivity, and significant in vivo activity indicate that CCT241736 is a bona fide clinical drug candidate for FLT3-ITD and TKD AML patients with resistance to current drugs.

Correction: Signalling involving MET and FAK supports cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High Proliferation Rate and a Compromised Spindle Assembly Checkpoint Confers Sensitivity to the MPS1 Inhibitor BOS172722 in Triple-Negative Breast Cancers.

BOS172722 (CCT289346) is a highly potent, selective, and orally bioavailable inhibitor of spindle assembly checkpoint kinase MPS1. BOS172722 treatment alone induces significant sensitization to death, particularly in highly proliferative triple-negative breast cancer (TNBC) cell lines with compromised spindle assembly checkpoint activity. BOS172722 synergizes with paclitaxel to induce gross chromosomal segregation defects caused by MPS1 inhibitor-mediated abrogation of the mitotic delay induced by paclitaxel treatment. In in vivo pharmacodynamic experiments, BOS172722 potently inhibits the spindle assembly checkpoint induced by paclitaxel in human tumor xenograft models of TNBC, as measured by inhibition of the phosphorylation of histone H3 and the phosphorylation of the MPS1 substrate, KNL1. This mechanistic synergy results in significant in vivo efficacy, with robust tumor regressions observed for the combination of BOS172722 and paclitaxel versus either agent alone in long-term efficacy studies in multiple human tumor xenograft TNBC models, including a patient-derived xenograft and a systemic metastasis model. The current target indication for BOS172722 is TNBC, based on their high sensitivity to MPS1 inhibition, the well-defined clinical patient population with high unmet need, and the synergy observed with paclitaxel.

Signalling involving MET and FAK supports cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases.

Deregulation of cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer; yet, inhibitors against these kinases are currently used only in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and the mechanisms that may undermine such dependency are poorly understood. Here, we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2 activation, involving as critical mechanistic events loss of the CDKI p21CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates the proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo. Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic.

ALK2 inhibitors display beneficial effects in preclinical models of ACVR1 mutant diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brainstem tumour, with a quarter of patients harbouring somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2. Despite being an amenable drug target, little has been done to-date to systematically evaluate the role of ACVR1 in DIPG, nor to screen currently available inhibitors in patient-derived tumour models. Here we show the dependence of DIPG cells on the mutant receptor, and the preclinical efficacy of two distinct chemotypes of ALK2 inhibitor in vitro and in vivo. We demonstrate the pyrazolo[1,5-a]pyrimidine LDN-193189 and the pyridine LDN-214117 to be orally bioavailable and well-tolerated, with good brain penetration. Treatment of immunodeprived mice bearing orthotopic xenografts of H3.3K27M, ACVR1R206H mutant HSJD-DIPG-007 cells with 25 mg/kg LDN-193189 or LDN-214117 for 28 days extended survival compared with vehicle controls. Development of ALK2 inhibitors with improved potency, selectivity and advantageous pharmacokinetic properties may play an important role in therapy for DIPG patients.

Binding to an Unusual Inactive Kinase Conformation by Highly Selective Inhibitors of Inositol-Requiring Enzyme 1α Kinase-Endoribonuclease.

A series of imidazo[1,2- b]pyridazin-8-amine kinase inhibitors were discovered to allosterically inhibit the endoribonuclease function of the dual kinase-endoribonuclease inositol-requiring enzyme 1α (IRE1α), a key component of the unfolded protein response in mammalian cells and a potential drug target in multiple human diseases. Inhibitor optimization gave compounds with high kinome selectivity that prevented endoplasmic reticulum stress-induced IRE1α oligomerization and phosphorylation, and inhibited endoribonuclease activity in human cells. X-ray crystallography showed the inhibitors to bind to a previously unreported and unusually disordered conformation of the IRE1α kinase domain that would be incompatible with back-to-back dimerization of the IRE1α protein and activation of the endoribonuclease function. These findings increase the repertoire of known IRE1α protein conformations and can guide the discovery of highly selective ligands for the IRE1α kinase site that allosterically inhibit the endoribonuclease.

Metabolic biomarkers of response to the AKT inhibitor MK-2206 in pre-clinical models of human colorectal and prostate carcinoma.

BACKGROUND: AKT is commonly overexpressed in tumours and plays an important role in the metabolic reprogramming of cancer. We have used magnetic resonance spectroscopy (MRS) to assess whether inhibition of AKT signalling would result in metabolic changes that could potentially be used as biomarkers to monitor response to AKT inhibition. METHODS: Cellular and metabolic effects of the allosteric AKT inhibitor MK-2206 were investigated in HT29 colon and PC3 prostate cancer cells and xenografts using flow cytometry, immunoblotting, immunohistology and MRS. RESULTS: In vitro treatment with MK-2206 inhibited AKT signalling and resulted in time-dependent alterations in glucose, glutamine and phospholipid metabolism. In vivo, MK-2206 resulted in inhibition of AKT signalling and tumour growth compared with vehicle-treated controls. In vivo MRS analysis of HT29 subcutaneous xenografts showed similar metabolic changes to those seen in vitro including decreases in the tCho/water ratio, tumour bioenergetic metabolites and changes in glutamine and glutathione metabolism. Similar phosphocholine changes compared to in vitro were confirmed in the clinically relevant orthotopic PC3 model. CONCLUSION: This MRS study suggests that choline metabolites detected in response to AKT inhibition are time and microenvironment-dependent, and may have potential as non-invasive biomarkers for monitoring response to AKT inhibitors in selected cancer types.

Introduction of a Methyl Group Curbs Metabolism of Pyrido[3,4- d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and Enables the Discovery of the Phase 1 Clinical Candidate N2-(2-Ethoxy-4-(4-methyl-4 H-1,2,4-triazol-3-yl)phenyl)-6-methyl- N8-neopentylpyrido[3,4- d]pyrimidine-2,8-diamine (BOS172722).

Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4- d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4- d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.

Correction to: An in vitro and in vivo study of the combination of the heat shock protein inhibitor 17-allylamino-17-demethoxygeldanamycin and carboplatin in human ovarian cancer models.

The corresponding author of this article has informed us of concerns about the immunoblots in Fig. 2 which were carried out in the collaborating laboratory of Professor Ann Jackman.

Evaluating Imaging Biomarkers of Acquired Resistance to Targeted EGFR Therapy in Xenograft Models of Human Head and Neck Squamous Cell Carcinoma.

Background: Overexpression of EGFR is a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). Patients with HNSCC who respond to EGFR-targeted tyrosine kinase inhibitors (TKIs) eventually develop acquired resistance. Strategies to identify HNSCC patients likely to benefit from EGFR-targeted therapies, together with biomarkers of treatment response, would have clinical value. Methods: Functional MRI and 18F-FDG PET were used to visualize and quantify imaging biomarkers associated with drug response within size-matched EGFR TKI-resistant CAL 27 (CALR) and sensitive (CALS) HNSCC xenografts in vivo, and pathological correlates sought. Results: Intrinsic susceptibility, oxygen-enhanced and dynamic contrast-enhanced MRI revealed significantly slower baseline R2∗ , lower hyperoxia-induced ΔR2∗ and volume transfer constant Ktrans in the CALR tumors which were associated with significantly lower Hoechst 33342 uptake and greater pimonidazole-adduct formation. There was no difference in oxygen-induced ΔR1 or water diffusivity between the CALR and CALS xenografts. PET revealed significantly higher relative uptake of 18F-FDG in the CALR cohort, which was associated with significantly greater Glut-1 expression. Conclusions: CALR xenografts established from HNSCC cells resistant to EGFR TKIs are more hypoxic, poorly perfused and glycolytic than sensitive CALS tumors. MRI combined with PET can be used to non-invasively assess HNSCC response/resistance to EGFR inhibition.

HSF1 Is Essential for Myeloma Cell Survival and A Promising Therapeutic Target.

Purpose: Myeloma is a plasma cell malignancy characterized by the overproduction of immunoglobulin, and is therefore susceptible to therapies targeting protein homeostasis. We hypothesized that heat shock factor 1 (HSF1) was an attractive therapeutic target for myeloma due to its direct regulation of transcriptional programs implicated in both protein homeostasis and the oncogenic phenotype. Here, we interrogate HSF1 as a therapeutic target in myeloma using bioinformatic, genetic, and pharmacologic means.Experimental Design: To assess the clinical relevance of HSF1, we analyzed publicly available patient myeloma gene expression datasets. Validation of this novel target was conducted in in vitro experiments using shRNA or inhibitors of the HSF1 pathway in human myeloma cell lines and primary cells as well as in in vivo human myeloma xenograft models.Results: Expression of HSF1 and its target genes were associated with poorer myeloma patient survival. ShRNA-mediated knockdown or pharmacologic inhibition of the HSF1 pathway with a novel chemical probe, CCT251236, or with KRIBB11, led to caspase-mediated cell death that was associated with an increase in EIF2α phosphorylation, CHOP expression and a decrease in overall protein synthesis. Importantly, both CCT251236 and KRIBB11 induced cytotoxicity in human myeloma cell lines and patient-derived primary myeloma cells with a therapeutic window over normal cells. Pharmacologic inhibition induced tumor growth inhibition and was well-tolerated in a human myeloma xenograft murine model with evidence of pharmacodynamic biomarker modulation.Conclusions: Taken together, our studies demonstrate the dependence of myeloma cells on HSF1 for survival and support the clinical evaluation of pharmacologic inhibitors of the HSF1 pathway in myeloma. Clin Cancer Res; 24(10); 2395-407. ©2018 AACRSee related commentary by Parekh, p. 2237.

Patient-derived organoids model treatment response of metastatic gastrointestinal cancers.

Patient-derived organoids (PDOs) have recently emerged as robust preclinical models; however, their potential to predict clinical outcomes in patients has remained unclear. We report on a living biobank of PDOs from metastatic, heavily pretreated colorectal and gastroesophageal cancer patients recruited in phase 1/2 clinical trials. Phenotypic and genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. Molecular profiling of tumor organoids was matched to drug-screening results, suggesting that PDOs could complement existing approaches in defining cancer vulnerabilities and improving treatment responses. We compared responses to anticancer agents ex vivo in organoids and PDO-based orthotopic mouse tumor xenograft models with the responses of the patients in clinical trials. Our data suggest that PDOs can recapitulate patient responses in the clinic and could be implemented in personalized medicine programs.

Functional imaging and circulating biomarkers of response to regorafenib in treatment-refractory metastatic colorectal cancer patients in a prospective phase II study.

OBJECTIVE: Regorafenib demonstrated efficacy in patients with metastatic colorectal cancer (mCRC). Lack of predictive biomarkers, potential toxicities and cost-effectiveness concerns highlight the unmet need for better patient selection. DESIGN: Patients with RAS mutant mCRC with biopsiable metastases were enrolled in this phase II trial. Dynamic contrast-enhanced (DCE) MRI was acquired pretreatment and at day 15 post-treatment. Median values of volume transfer constant (Ktrans), enhancing fraction (EF) and their product KEF (summarised median values of Ktrans× EF) were generated. Circulating tumour (ct) DNA was collected monthly until progressive disease and tested for clonal RAS mutations by digital-droplet PCR. Tumour vasculature (CD-31) was scored by immunohistochemistry on 70 sequential tissue biopsies. RESULTS: Twenty-seven patients with paired DCE-MRI scans were analysed. Median KEF decrease was 58.2%. Of the 23 patients with outcome data, >70% drop in KEF (6/23) was associated with higher disease control rate (p=0.048) measured by RECIST V. 1.1 at 2 months, improved progression-free survival (PFS) (HR 0.16 (95% CI 0.04 to 0.72), p=0.02), 4-month PFS (66.7% vs 23.5%) and overall survival (OS) (HR 0.08 (95% CI 0.01 to 0.63), p=0.02). KEF drop correlated with CD-31 reduction in sequential tissue biopsies (p=0.04). RAS mutant clones decay in ctDNA after 8 weeks of treatment was associated with better PFS (HR 0.21 (95% CI 0.06 to 0.71), p=0.01) and OS (HR 0.28 (95% CI 0.07-1.04), p=0.06). CONCLUSIONS: Combining DCE-MRI and ctDNA predicts duration of anti-angiogenic response to regorafenib and may improve patient management with potential health/economic implications.

MIR21 Drives Resistance to Heat Shock Protein 90 Inhibition in Cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinomas (CCA) are resistant to chemotherapy, so new therapeutic agents are needed. We performed a screen to identify small-molecule compounds that are active against CCAs. Levels of microRNA 21 (MIR21 or miRNA21) are increased in CCAs. We investigated whether miRNA21 mediates resistance of CCA cells and organoids to HSP90 inhibitors. METHODS: We performed a high-throughput screen of 484 small-molecule compounds to identify those that reduced viability of 6 human CCA cell lines. We tested the effects of HSP90 inhibitors on cells with disruption of the MIR21 gene, cells incubated with MIR21 inhibitors, and stable cell lines with inducible expression of MIR21. We obtained CCA biopsies from patients, cultured them as organoids (patient-derived organoids). We assessed their architecture, mutation and gene expression patterns, response to compounds in culture, and when grown as subcutaneous xenograft tumors in mice. RESULTS: Cells with IDH1 and PBRM1 mutations had the highest level of sensitivity to histone deacetylase inhibitors. HSP90 inhibitors were effective in all cell lines, irrespective of mutations. Sensitivity of cells to HSP90 inhibitors correlated inversely with baseline level of MIR21. Disruption of MIR21 increased cell sensitivity to HSP90 inhibitors. CCA cells that expressed transgenic MIR21 were more resistant to HSP90 inhibitors than cells transfected with control vectors; inactivation of MIR21 in these cells restored sensitivity to these agents. MIR21 was shown to target the DnaJ heat shock protein family (Hsp40) member B5 (DNAJB5). Transgenic expression of DNAJB5 in CCA cells that overexpressed MIR21 re-sensitized them to HSP90 inhibitors. Sensitivity of patient-derived organoids to HSP90 inhibitors, in culture and when grown as xenograft tumors in mice, depended on expression of miRNA21. CONCLUSIONS: miRNA21 appears to mediate resistance of CCA cells to HSP90 inhibitors by reducing levels of DNAJB5. HSP90 inhibitors might be developed for the treatment of CCA and miRNA21 might be a marker of sensitivity to these agents.