Large-scale Pan-cancer Cell Line Screening Identifies Actionable and Effective Drug Combinations.

Oncology drug combinations can improve therapeutic responses and increase treatment options for patients. The number of possible combinations is vast and responses can be context-specific. Systematic screens can identify clinically relevant, actionable combinations in defined patient subtypes. We present data for 109 anticancer drug combinations from AstraZeneca's oncology small molecule portfolio screened in 755 pan-cancer cell lines. Combinations were screened in a 7 × 7 concentration matrix, with more than 4 million measurements of sensitivity, producing an exceptionally data-rich resource. We implement a new approach using combination Emax (viability effect) and highest single agent (HSA) to assess combination benefit. We designed a clinical translatability workflow to identify combinations with clearly defined patient populations, rationale for tolerability based on tumor type and combination-specific "emergent" biomarkers, and exposures relevant to clinical doses. We describe three actionable combinations in defined cancer types, confirmed in vitro and in vivo, with a focus on hematologic cancers and apoptotic targets. SIGNIFICANCE: We present the largest cancer drug combination screen published to date with 7 × 7 concentration response matrices for 109 combinations in more than 750 cell lines, complemented by multi-omics predictors of response and identification of "emergent" combination biomarkers. We prioritize hits to optimize clinical translatability, and experimentally validate novel combination hypotheses. This article is featured in Selected Articles from This Issue, p. 695.

The ATR inhibitor ceralasertib potentiates cancer checkpoint immunotherapy by regulating the tumor microenvironment.

The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8+ T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8+ T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity.

SynBa: improved estimation of drug combination synergies with uncertainty quantification.

MOTIVATION: There exists a range of different quantification frameworks to estimate the synergistic effect of drug combinations. The diversity and disagreement in estimates make it challenging to determine which combinations from a large drug screening should be proceeded with. Furthermore, the lack of accurate uncertainty quantification for those estimates precludes the choice of optimal drug combinations based on the most favourable synergistic effect. RESULTS: In this work, we propose SynBa, a flexible Bayesian approach to estimate the uncertainty of the synergistic efficacy and potency of drug combinations, so that actionable decisions can be derived from the model outputs. The actionability is enabled by incorporating the Hill equation into SynBa, so that the parameters representing the potency and the efficacy can be preserved. Existing knowledge may be conveniently inserted due to the flexibility of the prior, as shown by the empirical Beta prior defined for the normalized maximal inhibition. Through experiments on large combination screenings and comparison against benchmark methods, we show that SynBa provides improved accuracy of dose-response predictions and better-calibrated uncertainty estimation for the parameters and the predictions. AVAILABILITY AND IMPLEMENTATION: The code for SynBa is available at https://github.com/HaotingZhang1/SynBa. The datasets are publicly available (DOI of DREAM: 10.7303/syn4231880; DOI of the NCI-ALMANAC subset: 10.5281/zenodo.4135059).

SRSF1-dependent inhibition of C9ORF72-repeat RNA nuclear export: genome-wide mechanisms for neuroprotection in amyotrophic lateral sclerosis.

BACKGROUND: Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. METHODS: Here, we investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of our gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. RESULTS: Our study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. We also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1-3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. CONCLUSIONS: Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.

Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions.

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.

Translating SOD1 Gene Silencing toward the Clinic: A Highly Efficacious, Off-Target-free, and Biomarker-Supported Strategy for fALS.

Of familial amyotrophic lateral sclerosis (fALS) cases, 20% are caused by mutations in the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of the therapeutic potential of RNAi for the treatment of SOD1-ALS patients requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88%, and prolonged the median survival of SOD1G93A mice by up to 42%. To our knowledge, this is the first report to demonstrate no significant off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of biological activity for translation into human clinical trials.

The expression of oxidative stress response genes is modulated by a combination of resveratrol and N-acetylcysteine to ameliorate ototoxicity in the rat cochlea.

Aminoglycoside antibiotics are used widely in medicine despite their ototoxic side-effects. Oxidative stress and inflammation are key mechanisms determining the extent and severity of the damage. Here we evaluate the protective effect of a treatment with resveratrol plus N-acetylcysteine on the ototoxic actions of kanamycin and furosemide in the rat. Resveratrol (10 mg/kg) and N-acetylcysteine (400 mg/kg) were administered together to Wistar rats on 5 consecutive days. The second day, a concentrated solution of kanamycin and furosemide was placed on the round window to induce ototoxicity. Hearing was assessed by recording auditory brainstem responses before and 5, 16 and 23 days after the beginning of the treatment. Cochlear samples were taken at day 5 (end of the treatment) and at day 23, and targeted PCR arrays or RT-qPCR were performed to analyze oxidative balance and inflammation related genes, respectively. In addition, the cytoarchitecture and the presence of apoptosis, oxidative stress and inflammation markers were evaluated in cochlear sections. Results indicate that administration of resveratrol plus N-acetylcysteine reduced the threshold shifts induced by ototoxic drugs at high frequencies (≈10 dB), although this protective effect fades after the cessation of the treatment. Gene expression analysis showed that the treatment modulated the expression of genes involved in the cellular oxidative (Gpx1, Sod1, Ccs and Noxa1) and inflammatory (Il1b, Il4, Mpo and Ncf) responses to injury. Thus, co-administration of resveratrol and NAC, routinely used individually in patients, could reduce the ototoxic secondary effects of aminoglycosides.

Serum miRNAs miR-206, 143-3p and 374b-5p as potential biomarkers for amyotrophic lateral sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative condition characterized by loss of motor neurones and progressive muscle wasting. There is no diagnostic test for ALS therefore robust biomarkers would not only be valuable for diagnosis, but also for the classification of disease subtypes, monitoring responses to drugs and tracking disease progression. As regulators of gene expression, microRNAs (miRNAs) are increasingly used for diagnostic and prognostic purposes in various disease states with increasing exploration in neurodegenerative disorders. We hypothesize that circulating blood-based miRNAs will serve as biomarkers and use miRNA profiling to determine miRNA signatures from the serum of sporadic ALS patients compared to healthy controls and patients with diseases that mimic ALS. A number of differentially expressed miRNAs were identified in each set of patient comparisons. Validation in an additional patient cohort showed that miR-206 and miR-143-3p were increased and miR-374b-5p was decreased compared to controls. A continued change in miRNA expression persisted during disease progression indicating the potential use of these particular miRNAs as longitudinal biomarkers in ALS.

Multipotency of Adult Hippocampal NSCs In Vivo Is Restricted by Drosha/NFIB.

Adult neural stem cells (NSCs) are defined by their inherent capacity to self-renew and give rise to neurons, astrocytes, and oligodendrocytes. In vivo, however, hippocampal NSCs do not generate oligodendrocytes for reasons that have remained enigmatic. Here, we report that deletion of Drosha in adult dentate gyrus NSCs activates oligodendrogenesis and reduces neurogenesis at the expense of gliogenesis. We further find that Drosha directly targets NFIB to repress its expression independently of Dicer and microRNAs. Knockdown of NFIB in Drosha-deficient hippocampal NSCs restores neurogenesis, suggesting that the Drosha/NFIB mechanism robustly prevents oligodendrocyte fate acquisition in vivo. Taken together, our findings establish that adult hippocampal NSCs inherently possess multilineage potential but that Drosha functions as a molecular barrier preventing oligodendrogenesis.

Comparative gene expression study of the vestibular organ of the Igf1 deficient mouse using whole-transcript arrays.

The auditory and vestibular organs form the inner ear and have a common developmental origin. Insulin like growth factor 1 (IGF-1) has a central role in the development of the cochlea and maintenance of hearing. Its deficiency causes sensorineural hearing loss in man and mice. During chicken early development, IGF-1 modulates neurogenesis of the cochleovestibular ganglion but no further studies have been conducted to explore the potential role of IGF-1 in the vestibular system. In this study we have compared the whole transcriptome of the vestibular organ from wild type and Igf1(-/-) mice at different developmental and postnatal times. RNA was prepared from E18.5, P15 and P90 vestibular organs of Igf1(-/-) and Igf1(+/+) mice and the transcriptome analysed in triplicates using Affymetrix(®) Mouse Gene 1.1 ST Array Plates. These plates are whole-transcript arrays that include probes to measure both messenger (mRNA) and long intergenic non-coding RNA transcripts (lincRNA), with a coverage of over 28 thousand coding transcripts and over 7 thousands non-coding transcripts. Given the complexity of the data we used two different methods VSN-RMA and mmBGX to analyse and compare the data. This is to better evaluate the number of false positives and to quantify uncertainty of low signals. We identified a number of differentially expressed genes that we described using functional analysis and validated using RT-qPCR. The morphology of the vestibular organ did not show differences between genotypes and no evident alterations were observed in the vestibular sensory areas of the null mice. However, well-defined cellular alterations were found in the vestibular neurons with respect their number and size. Although these mice did not show a dramatic vestibular phenotype, we conducted a functional analysis on differentially expressed genes between genotypes and across time. This was with the aim to identify new pathways that are involved in the development of the vestibular organ as well as pathways that maybe affected by the lack of IGF-1 and be associated to the morphological changes of the vestibular neurons that we observed in the Igf1(-/-) mice.

Gene expression signatures in motor neurone disease fibroblasts reveal dysregulation of metabolism, hypoxia-response and RNA processing functions.

AIMS: Amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS) are two syndromic variants within the motor neurone disease spectrum. As PLS and most ALS cases are sporadic (SALS), this limits the availability of cellular models for investigating pathogenic mechanisms and therapeutic targets. The aim of this study was to use gene expression profiling to evaluate fibroblasts as cellular models for SALS and PLS, to establish whether dysregulated biological processes recapitulate those seen in the central nervous system and to elucidate pathways that distinguish the clinically defined variants of SALS and PLS. METHODS: Microarray analysis was performed on fibroblast RNA and differentially expressed genes identified. Genes in enriched biological pathways were validated by quantitative PCR and functional assays performed to establish the effect of altered RNA levels on the cellular processes. RESULTS: Gene expression profiling demonstrated that whilst there were many differentially expressed genes in common between SALS and PLS fibroblasts, there were many more expressed specifically in the SALS fibroblasts, including those involved in RNA processing and the stress response. Functional analysis of the fibroblasts confirmed a significant decrease in miRNA production and a reduced response to hypoxia in SALS fibroblasts. Furthermore, metabolic gene changes seen in SALS, many of which were also evident in PLS fibroblasts, resulted in dysfunctional cellular respiration. CONCLUSIONS: The data demonstrate that fibroblasts can act as cellular models for ALS and PLS, by establishing the transcriptional changes in known pathogenic pathways that confer subsequent functional effects and potentially highlight targets for therapeutic intervention.

TigarB causes mitochondrial dysfunction and neuronal loss in PINK1 deficiency.

OBJECTIVE: Loss of function mutations in PINK1 typically lead to early onset Parkinson disease (PD). Zebrafish (Danio rerio) are emerging as a powerful new vertebrate model to study neurodegenerative diseases. We used a pink1 mutant (pink(-/-) ) zebrafish line with a premature stop mutation (Y431*) in the PINK1 kinase domain to identify molecular mechanisms leading to mitochondrial dysfunction and loss of dopaminergic neurons in PINK1 deficiency. METHODS: The effect of PINK1 deficiency on the number of dopaminergic neurons, mitochondrial function, and morphology was assessed in both zebrafish embryos and adults. Genome-wide gene expression studies were undertaken to identify novel pathogenic mechanisms. Functional experiments were carried out to further investigate the effect of PINK1 deficiency on early neurodevelopmental mechanisms and microglial activation. RESULTS: PINK1 deficiency results in loss of dopaminergic neurons as well as early impairment of mitochondrial function and morphology in Danio rerio. Expression of TigarB, the zebrafish orthologue of the human, TP53-induced glycolysis and apoptosis regulator TIGAR, was markedly increased in pink(-/-) larvae. Antisense-mediated inactivation of TigarB gave rise to complete normalization of mitochondrial function, with resulting rescue of dopaminergic neurons in pink(-/-) larvae. There was also marked microglial activation in pink(-/-) larvae, but depletion of microglia failed to rescue the dopaminergic neuron loss, arguing against microglial activation being a key factor in the pathogenesis. INTERPRETATION: Pink1(-/-) zebrafish are the first vertebrate model of PINK1 deficiency with loss of dopaminergic neurons. Our study also identifies TIGAR as a promising novel target for disease-modifying therapy in PINK1-related PD.

Restoration of auditory evoked responses by human ES-cell-derived otic progenitors.

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells, is responsible for a substantial proportion of patients with hearing impairment. Although the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss, as poor innervation limits the prospective performance of an implant. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair-cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory-evoked response thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

Drosha regulates neurogenesis by controlling neurogenin 2 expression independent of microRNAs.

Temporal regulation of embryonic neurogenesis is controlled by hypostable transcription factors. The mechanism of the process is unclear. Here we show that the RNase III Drosha and DGCR8 (also known as Pasha), key components of the microRNA (miRNA) microprocessor, have important functions in mouse neurogenesis. Loss of microprocessor in forebrain neural progenitors resulted in a loss of stem cell character and precocious differentiation whereas Dicer deficiency did not. Drosha negatively regulated expression of the transcription factors Neurogenin 2 (Ngn2) and NeuroD1 whereas forced Ngn2 expression phenocopied the loss of Drosha. Neurog2 mRNA contains evolutionarily conserved hairpins with similarities to pri-miRNAs, and associates with the microprocessor in neural progenitors. We uncovered a Drosha-dependent destabilization of Neurog2 mRNAs consistent with microprocessor cleavage at hairpins. Our findings implicate direct and miRNA-independent destabilization of proneural mRNAs by the microprocessor, which facilitates neural stem cell (NSC) maintenance by blocking accumulation of differentiation and determination factors.

Prolyl hydroxylase 3 (PHD3) is essential for hypoxic regulation of neutrophilic inflammation in humans and mice.

The regulation of neutrophil lifespan by induction of apoptosis is critical for maintaining an effective host response and preventing excessive inflammation. The hypoxia-inducible factor (HIF) oxygen-sensing pathway has a major effect on the susceptibility of neutrophils to apoptosis, with a marked delay in cell death observed under hypoxic conditions. HIF expression and transcriptional activity are regulated by the oxygen-sensitive prolyl hydroxylases (PHD1-3), but the role of PHDs in neutrophil survival is unclear. We examined PHD expression in human neutrophils and found that PHD3 was strongly induced in response to hypoxia and inflammatory stimuli in vitro and in vivo. Using neutrophils from mice deficient in Phd3, we demonstrated a unique role for Phd3 in prolonging neutrophil survival during hypoxia, distinct from other hypoxia-associated changes in neutrophil function and metabolic activity. Moreover, this selective defect in neutrophil survival occurred in the presence of preserved HIF transcriptional activity but was associated with upregulation of the proapoptotic mediator Siva1 and loss of its binding target Bcl-xL. In vivo, using an acute lung injury model, we observed increased levels of neutrophil apoptosis and clearance in Phd3-deficient mice compared with WT controls. We also observed reduced neutrophilic inflammation in an acute mouse model of colitis. These data support what we believe to be a novel function for PHD3 in regulating neutrophil survival in hypoxia and may enable the development of new therapeutics for inflammatory disease.

Regulation of neutrophil senescence by microRNAs.

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease.

Study protocol: a randomised controlled trial investigating the effect of exercise training on peripheral blood gene expression in patients with stable angina.

BACKGROUND: Exercise training has been shown to reduce angina and promote collateral vessel development in patients with coronary artery disease. However, the mechanism whereby exercise exerts these beneficial effects is unclear. There has been increasing interest in the use of whole genome peripheral blood gene expression in a wide range of conditions to attempt to identify both novel mechanisms of disease and transcriptional biomarkers. This protocol describes a study in which we will assess the effect of a structured exercise programme on peripheral blood gene expression in patients with stable angina, and correlate this with changes in angina level, anxiety, depression, and exercise capacity. METHODS/DESIGN: Sixty patients with stable angina will be recruited and randomised 1:1 to exercise training or conventional care. Patients randomised to exercise training will attend an exercise physiology laboratory up to three times weekly for supervised aerobic interval training sessions of one hour in total duration. Patients will undergo assessments of angina, anxiety, depression, and peripheral blood gene expression at baseline, after six and twelve weeks of training, and twelve weeks after formal exercise training ceases. DISCUSSION: This study will provide comprehensive data on the effect of exercise training on peripheral blood gene expression in patients with angina. By correlating this with improvement in angina status we will identify candidate peripheral blood transcriptional markers predictive of improvements in angina level in response to exercise training. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01147952.

Transcriptional targets of Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour formation.

Although many signal transduction pathways have been implicated in the development of human disease, the identification of pathway targets and the biological processes that mediate disease progression remains challenging. One such disease-related pathway is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cascade whose constitutive misactivation by the JAK2 V617F mutation underlies most human myeloproliferative disorders. Here, we use transcript profiling of Drosophila haemocyte-like cells to identify JAK/STAT target genes, combined with an in vivo model for JAK-induced blood cell overproliferation, to identify the main effectors required for haematopoietic tumour development. The identified human homologues of the Drosophila effectors were tested for potential V617F-mediated transcriptional regulation in human HeLa cells and compared with small interfering RNA-derived data, quantify their role in regulating the proliferation of cancer-derived cell lines. Such an inter-species approach is an effective way to identify factors with conserved functions that might be central to human disease.

RNA microarray analysis in prenatal mouse cochlea reveals novel IGF-I target genes: implication of MEF2 and FOXM1 transcription factors.

BACKGROUND: Insulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness. METHODOLOGY/PRINCIPAL FINDINGS: A combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1(-/-) null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1(-/-) and Igf1(+/+) transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1(+/+) mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27(Kip1). CONCLUSIONS/SIGNIFICANCE: We have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I.