Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy.

BACKGROUND: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. METHODS: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. RESULTS: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. CONCLUSIONS: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850.

A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.

There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.

Investigating intracranial tumour growth patterns with multiparametric MRI incorporating Gd-DTPA and USPIO-enhanced imaging.

High grade and metastatic brain tumours exhibit considerable spatial variations in proliferation, angiogenesis, invasion, necrosis and oedema. Vascular heterogeneity arising from vascular co-option in regions of invasive growth (in which the blood-brain barrier remains intact) and neoangiogenesis is a major challenge faced in the assessment of brain tumours by conventional MRI. A multiparametric MRI approach, incorporating native measurements and both Gd-DTPA (Magnevist) and ultrasmall superparamagnetic iron oxide (P904)-enhanced imaging, was used in combination with histogram and unsupervised cluster analysis using a k-means algorithm to examine the spatial distribution of vascular parameters, water diffusion characteristics and invasion in intracranially propagated rat RG2 gliomas and human MDA-MB-231 LM2-4 breast adenocarcinomas in mice. Both tumour models presented with higher ΔR1 (the change in transverse relaxation rate R1 induced by Gd-DTPA), fractional blood volume (fBV) and apparent diffusion coefficient than uninvolved regions of the brain. MDA-MB-231 LM2-4 tumours were less densely cellular than RG2 tumours and exhibited substantial local invasion, associated with oedema, whereas invasion in RG2 tumours was minimal. These additional features were reflected in the more heterogeneous appearance of MDA-MB-231 LM2-4 tumours on T2 -weighted images and maps of functional MRI parameters. Unsupervised cluster analysis separated subregions with distinct functional properties; areas with a low fBV and relatively impermeable blood vessels (low ΔR1 ) were predominantly located at the tumour margins, regions of MDA-MB-231 LM2-4 tumours with relatively high levels of water diffusion and low vascular permeability and/or fBV corresponded to histologically identified regions of invasion and oedema, and areas of mismatch between vascular permeability and blood volume were identified. We demonstrate that dual contrast MRI and evaluation of tissue diffusion properties, coupled with cluster analysis, allows for the assessment of heterogeneity within invasive brain tumours and the designation of functionally diverse subregions that may provide more informative predictive biomarkers.

Dependence of Wilms tumor cells on signaling through insulin-like growth factor 1 in an orthotopic xenograft model targetable by specific receptor inhibition.

We have previously demonstrated an increased DNA copy number and expression of IGF1R to be associated with poor outcome in Wilms tumors. We have now tested whether inhibiting this receptor may be a useful therapeutic strategy by using a panel of Wilms tumor cell lines. Both genetic and pharmacological targeting resulted in inhibition of downstream signaling through PI3 and MAP kinases, G(1) cell cycle arrest, and cell death, with drug efficacy dependent on the levels of phosphorylated IGF1R. These effects were further associated with specific gene expression signatures reflecting pathway inhibition, and conferred synergistic chemosensitisation to doxorubicin and topotecan. In the in vivo setting, s.c. xenografts of WiT49 cells resembled malignant rhabdoid tumors rather than Wilms tumors. Treatment with an IGF1R inhibitor (NVP-AEW541) showed no discernable antitumor activity and no downstream pathway inactivation. By contrast, Wilms tumor cells established orthotopically within the kidney were histologically accurate and exhibited significantly elevated insulin-like growth factor-mediated signaling, and growth was significantly reduced on treatment with NVP-AEW541 in parallel with signaling pathway ablation. As a result of the paracrine effects of enhanced IGF2 expression in Wilms tumor, this disease may be acutely dependent on signaling through the IGF1 receptor, and thus treatment strategies aimed at its inhibition may be useful in the clinic. Such efficacy may be missed if only standard ectopic models are considered as a result of an imperfect recapitulation of the specific tumor microenvironment.

Discovery of an In Vivo Chemical Probe for BCL6 Inhibition by Optimization of Tricyclic Quinolinones.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.

Non-invasive in vivo imaging of vessel calibre in orthotopic prostate tumour xenografts.

Susceptibility contrast magnetic resonance imaging (MRI), utilising ultrasmall superparamagnetic iron oxide (USPIO) particles, was evaluated for the quantitation of vessel size index (Rv, µm), a weighted average measure of tumour blood vessel calibre, and fractional tumour blood volume (fBV, %), in orthotopically propagated murine PC3 prostate tumour xenografts. Tumour vascular architecture was assessed in vivo by MRI prior to and 24 hr after treatment with 200 mg/kg of the vascular disrupting agent ZD6126. A Bayesian hierarchical model (BHM) was used to reduce the uncertainty associated with quantitation of Rv and fBV. Quantitative histological analyses of the uptake of Hoechst 33342 for perfused vasculature, and haematoxylin and eosin staining for necrosis, were also performed to qualify the MRI data. A relatively large median Rv of 40.3 µm (90% confidence interval (CI90) = 37.4, 44.0 µm) and a high fBV of 5.4% (CI90 = 5.3, 5.5%) were determined in control tumours, which agreed with histologically determined vessel size index. Treatment with ZD6126 significantly (p < 0.01) reduced tumour Rv (34.2 µm, CI90 = 31.2, 38.0 µm) and fBV (3.9%, CI90 = 3.8, 4.1%), which were validated against histologically determined significant reductions in perfusion and vessel size, and increased necrosis. Together these data (i) highlight the use of a BHM to optimise the inferential power available from susceptibility contrast MRI data, (ii) provide strong evaluation and qualification of R(v) and fBV as non-invasive imaging biomarkers of tumour vascular morphology, (iii) reveal the presence of a different vascular phenotype and (iv) demonstrate that ZD6126 exhibits good anti-vascular activity against orthotopic prostate tumours.

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.

FGF7-FGFR2 autocrine signaling increases growth and chemoresistance of fusion-positive rhabdomyosarcomas.

Rhabdomyosarcomas are aggressive pediatric soft-tissue sarcomas and include high-risk PAX3-FOXO1 fusion-gene-positive cases. Fibroblast growth factor receptor 4 (FGFR4) is known to contribute to rhabdomyosarcoma progression; here, we sought to investigate the involvement and potential for therapeutic targeting of other FGFRs in this disease. Cell-based screening of FGFR inhibitors with potential for clinical repurposing (NVP-BGJ398, nintedanib, dovitinib, and ponatinib) revealed greater sensitivity of fusion-gene-positive versus fusion-gene-negative rhabdomyosarcoma cell lines and was shown to be correlated with high expression of FGFR2 and its specific ligand, FGF7. Furthermore, patient samples exhibit higher mRNA levels of FGFR2 and FGF7 in fusion-gene-positive versus fusion-gene-negative rhabdomyosarcomas. Sustained intracellular mitogen-activated protein kinase (MAPK) activity and FGF7 secretion into culture media during serum starvation of PAX3-FOXO1 rhabdomyosarcoma cells together with decreased cell viability after genetic silencing of FGFR2 or FGF7 was in keeping with a novel FGF7-FGFR2 autocrine loop. FGFR inhibition with NVP-BGJ398 reduced viability and was synergistic with SN38, the active metabolite of irinotecan. In vivo, NVP-BGJ398 abrogated xenograft growth and warrants further investigation in combination with irinotecan as a therapeutic strategy for fusion-gene-positive rhabdomyosarcomas.

Improved Binding Affinity and Pharmacokinetics Enable Sustained Degradation of BCL6 In Vivo.

The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo. We observed a sharp degradation SAR, where subtle structural changes conveyed the ability to induce degradation of BCL6. CCT373566 showed modest in vivo efficacy in a lymphoma xenograft mouse model following oral dosing.

Bayesian estimation of changes in transverse relaxation rates.

Although the biasing of R(2)* estimates by assuming magnitude MR data to be normally distributed has been described, the effect on changes in R(2)* (DeltaR(2)*), such as induced by a paramagnetic contrast agent, has not been reported. In this study, two versions of a novel Bayesian maximum a posteriori approach for estimating DeltaR(2)* are described and evaluated: one that assumes normally distributed data and the other, Rice-distributed data. The approach enables the robust, voxelwise determination of the uncertainty in DeltaR(2)* estimates and provides a useful statistical framework for quantifying the probability that a pixel has been significantly enhanced. This technique was evaluated in vivo, using ultrasmall superparamagnetic iron oxide particles in orthotopic murine prostate tumors. It is shown that assuming magnitude data to be normally distributed causes DeltaR(2)* to be underestimated when signal-to-noise ratio is modest. However, the biasing effect is less than is found in R(2)* estimates, implying that the simplifying assumption of normally distributed noise is more justifiable when evaluating DeltaR(2)* compared with when evaluating precontrast R(2)* values.

Plexin-B1 mutations in prostate cancer.

Semaphorins are a large class of secreted or membrane-associated proteins that act as chemotactic cues for cell movement via their transmembrane receptors, plexins. We hypothesized that the function of the semaphorin signaling pathway in the control of cell migration could be harnessed by cancer cells during invasion and metastasis. We now report 13 somatic missense mutations in the cytoplasmic domain of the Plexin-B1 gene. Mutations were found in 89% (8 of 9) of prostate cancer bone metastases, in 41% (7 of 17) of lymph node metastases, and in 46% (41 of 89) of primary cancers. Forty percent of prostate cancers contained the same mutation. Overexpression of the Plexin-B1 protein was found in the majority of primary tumors. The mutations hinder Rac and R-Ras binding and R-RasGAP activity, resulting in an increase in cell motility, invasion, adhesion, and lamellipodia extension. These results identify a key role for Plexin-B1 and the semaphorin signaling pathway it mediates in prostate cancer.

Identification of ovarian high-grade serous carcinoma cell lines that show estrogen-sensitive growth as xenografts in immunocompromised mice.

Ovarian cancer remains a significant challenge in women worldwide. Tumors of the high-grade serous carcinoma (HGSC) type represent the most common form of the disease. Development of new therapies for HGSC has been hampered by a paucity of preclinical models in which new drugs could be tested for target engagement and anti-tumor efficacy. Here, we systematically assessed in vivo growth of ovarian cancer cells, including six validated HGSC cell lines, in highly immunocompromised NSG mice by varying the injection site. We found that, with the exception of OVCAR3, HGSC cell lines COV318, COV362, KURAMOCHI, OVCAR4, and OVSAHO, generally demonstrate poor growth as either subcutaneous or intraperitoneal xenografts. Intrabursal injections performed with KURAMOCHI and COV362 cells did not improve tumor growth in vivo. Additional analysis revealed that OVSAHO and COV362 express moderate levels of estrogen receptor (ERα), which translated into improved growth of xenografts in the presence of 17ß-Estradiol. Surprisingly, we also found that the growth of the widely used non-HGSC ovarian cell line SKOV3 could be significantly improved by estrogen supplementation. By describing successful establishment of estrogen-sensitive HGSC xenograft models, OVSAHO and COV362, this work will enable testing of novel therapies for this aggressive form of ovarian cancer.

Theranostic Attributes of Acoustic Cluster Therapy and Its Use for Enhancing the Effectiveness of Liposomal Doxorubicin Treatment of Human Triple Negative Breast Cancer in Mice.

INTRODUCTION: Acoustic cluster therapy (ACT) comprises co-administration of a formulation containing microbubble/microdroplet clusters (PS101), together with a regular medicinal drug (e.g., a chemotherapeutic) and local ultrasound (US) insonation of the targeted pathological tissue (e.g., the tumor). PS101 is confined to the vascular compartment and, when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase-shift to produce bubbles with a median diameter of 22 µm when unconstrained by the capillary wall. In vivo these bubbles transiently lodge in the tumor's microvasculature. Low frequency ultrasound (300 kHz) at a low mechanical index (MI = 0.15) is then applied to drive oscillations of the deposited ACT bubbles to induce a range of biomechanical effects that locally enhance extravasation, distribution, and uptake of the co-administered drug, significantly increasing its therapeutic efficacy. METHODS: In this study we investigated the therapeutic efficacy of ACT with liposomal doxorubicin for the treatment of triple negative breast cancer using orthotopic human tumor xenografts (MDA-MB-231-H.luc) in athymic mice (ICR-NCr-Foxn1nu). Doxil® (6 mg/kg, i.v.) was administered at days 0 and 21, each time immediately followed by three sequential ACT (20 ml/kg PS101) treatment procedures (n = 7-10). B-mode and nonlinear ultrasound images acquired during the activation phase were correlated to the therapeutic efficacy. RESULTS: Results show that combination with ACT induces a strong increase in the therapeutic efficacy of Doxil®, with 63% of animals in complete, stable remission at end of study, vs. 10% for Doxil® alone (p < 0.02). A significant positive correlation (p < 0.004) was found between B-mode contrast enhancement during ACT activation and therapy response. These observations indicate that ACT may also be used as a theranostic agent and that ultrasound contrast enhancement during or before ACT treatment may be employed as a biomarker of therapeutic response during clinical use.

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.

Achieving In Vivo Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

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.

In vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol class of heat shock protein 90 inhibitors.

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.

Therapeutic Dose Response of Acoustic Cluster Therapy in Combination With Irinotecan for the Treatment of Human Colon Cancer in Mice.

Introduction: Acoustic Cluster Therapy (ACT) comprises coadministration of a formulation containing microbubble-microdroplet clusters (PS101) together with a regular medicinal drug and local ultrasound (US) insonation of the targeted pathological tissue. PS101 is confined to the vascular compartment and when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase shift to produce bubbles with a median diameter of 22 µm. Low frequency, low mechanical index US is then applied to drive oscillations of the deposited ACT bubbles to induce biomechanical effects that locally enhance extravasation, distribution, and uptake of the coadministered drug, significantly increasing its therapeutic efficacy. Methods: The therapeutic efficacy of ACT with irinotecan (60 mg/kg i.p.) was investigated using three treatment sessions given on day 0, 7, and 14 on subcutaneous human colorectal adenocarcinoma xenografts in mice. Treatment was performed with three back-to-back PS101+US administrations per session with PS101 doses ranging from 0.40-2.00 ml PS101/kg body weight (n = 8-15). To induce the phase shift, 45 s of US at 8 MHz at an MI of 0.30 was applied using a diagnostic US system; low frequency exposure consisted of 1 or 5 min at 500 kHz with an MI of 0.20. Results: ACT with irinotecan induced a strong, dose dependent increase in the therapeutic effect (R2 = 0.95). When compared to irinotecan alone, at the highest dose investigated, combination treatment induced a reduction in average normalized tumour volume from 14.6 (irinotecan), to 5.4 (ACT with irinotecan, p = 0.002) on day 27. Median survival increased from 34 days (irinotecan) to 54 (ACT with irinotecan, p = 0.002). Additionally, ACT with irinotecan induced an increase in the fraction of complete responders; from 7% to 26%. There was no significant difference in the therapeutic efficacy whether the low frequency US lasted 1 or 5 min. Furthermore, there was no significant difference between the enhancement observed in the efficacy of ACT with irinotecan when PS101+US was administered before or after irinotecan. An increase in early dropouts was observed at higher PS101 doses. Both mean tumour volume (on day 27) and median survival indicate that the PS101 dose response was linear in the range investigated.

Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer.

Despite showing clinical activity in BRAF-mutant melanoma, the MEK inhibitor (MEKi) trametinib has failed to show clinical benefit in KRAS-mutant colorectal cancer. To identify mechanisms of resistance to MEKi, we employed a pharmacogenomic analysis of MEKi-sensitive versus MEKi-resistant colorectal cancer cell lines. Strikingly, interferon- and inflammatory-related gene sets were enriched in cell lines exhibiting intrinsic and acquired resistance to MEK inhibition. The bromodomain inhibitor JQ1 suppressed interferon-stimulated gene (ISG) expression and in combination with MEK inhibitors displayed synergistic effects and induced apoptosis in MEKi-resistant colorectal cancer cell lines. ISG expression was confirmed in patient-derived organoid models, which displayed resistance to trametinib and were resensitized by JQ1 co-treatment. In in vivo models of colorectal cancer, combination treatment significantly suppressed tumor growth. Our findings provide a novel explanation for the limited response to MEK inhibitors in KRAS-mutant colorectal cancer, known for its inflammatory nature. Moreover, the high expression of ISGs was associated with significantly reduced survival of colorectal cancer patients. Excitingly, we have identified novel therapeutic opportunities to overcome intrinsic and acquired resistance to MEK inhibition in colorectal cancer.

Correction: Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer.

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