SGLT2 inhibition improves PI3Kα inhibitor-induced hyperglycemia: findings from preclinical animal models and from patients in the BYLieve and SOLAR-1 trials.

PURPOSE: Alpelisib plus fulvestrant demonstrated a significant progression-free survival benefit versus fulvestrant in patients with PIK3CA-mutated HR+ /HER2- advanced breast cancer (ABC) (SOLAR-1). Hyperglycemia, an on-target adverse effect of PI3Kα inhibition, can lead to dose modifications, potentially impacting alpelisib efficacy. We report data from preclinical models and two clinical trials (SOLAR-1 and BYLieve) on Sodium glucose cotransporter 2 inhibitor (SGLT2i) use to improve PI3Kα inhibitor-associated hyperglycemia. METHODS: Healthy Brown Norway (BN), mild diabetic Zucker diabetic fatty (ZDF), and Rat1-myr-p110α/HBRX3077 tumor-bearing nude rats treated with alpelisib were analyzed for glucose and insulin control with metformin and dapagliflozin (SGLT2i) and alpelisib efficacy. Hyperglycemia adverse events (AEs) were compared between patients receiving SGLT2i with alpelisib (n = 19) and a propensity score-matched cohort not receiving SGLT2i (n = 74) in both trials. RESULTS: Dapagliflozin and metformin in BN and ZDF rats treated with alpelisib normalized blood glucose and reduced insulin levels. No signs of ketosis or drug-drug interaction were observed when metformin and dapagliflozin was administered with alpelisib. Alpelisib antitumor efficacy was maintained when used with dapagliflozin in tumor-bearing rats. Compared with a matched set of patients without SGLT2i, patients receiving SGLT2i had 4.9 and 6.4 times lower rates of grade ≥ 3 hyperglycemia AEs and hyperglycemia AEs resulting in alpelisib dose adjustments, interruptions, or withdrawals, respectively, and a relative reduction in risk of experiencing these AEs (70.6% and 35.7%). CONCLUSION: These data suggest adding an SGLT2i can effectively manage hyperglycemia, resulting in fewer alpelisib dose modifications and discontinuations in patients with PIK3CA-mutated HR+ /HER2- ABC (SOLAR-1: NCT02437318; BYLieve: NCT03056755).

INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

Integrated CRISPR screening and drug profiling identifies combination opportunities for EGFR, ALK, and BRAF/MEK inhibitors.

Anti-tumor efficacy of targeted therapies is variable across patients and cancer types. Even in patients with initial deep response, tumors are typically not eradicated and eventually relapse. To address these challenges, we present a systematic screen for targets that limit the anti-tumor efficacy of EGFR and ALK inhibitors in non-small cell lung cancer and BRAF/MEK inhibitors in colorectal cancer. Our approach includes genome-wide CRISPR screens with or without drugs targeting the oncogenic driver ("anchor therapy"), and large-scale pairwise combination screens of anchor therapies with 351 other drugs. Interestingly, targeting of a small number of genes, including MCL1, BCL2L1, and YAP1, sensitizes multiple cell lines to the respective anchor therapy. Data from drug combination screens with EGF816 and ceritinib indicate that dasatinib and agents disrupting microtubules act synergistically across many cell lines. Finally, we show that a higher-order-combination screen with 26 selected drugs in two resistant EGFR-mutant lung cancer cell lines identified active triplet combinations.

Identification of NVP-CLR457 as an Orally Bioavailable Non-CNS-Penetrant pan-Class IA Phosphoinositol-3-Kinase Inhibitor.

Balanced pan-class I phosphoinositide 3-kinase inhibition as an approach to cancer treatment offers the prospect of treating a broad range of tumor types and/or a way to achieve greater efficacy with a single inhibitor. Taking buparlisib as the starting point, the balanced pan-class I PI3K inhibitor 40 (NVP-CLR457) was identified with what was considered to be a best-in-class profile. Key to the optimization to achieve this profile was eliminating a microtubule stabilizing off-target activity, balancing the pan-class I PI3K inhibition profile, minimizing CNS penetration, and developing an amorphous solid dispersion formulation. A rationale for the poor tolerability profile of 40 in a clinical study is discussed.

Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.

3-dimensional printing for the diagnosis of left ventricular outflow tract obstruction after mitral valve replacement.

The objective of this study was to evaluate the use of the generation of 3D models and 3D prints of complex cases for physicians at the example of an intricate left ventricular outflow tract obstruction (LVOTO). LVOTO is a known complication of mitral valve surgery. A 38-year-old female patient with increasing dyspnoea after mitral valve replacement was referred to our centre. Echocardiography showed a strut of the bioprosthetic heart valve protruding into the left ventricular outflow tract. However, the diagnosis of a LVOTO was difficult based on echocardiography alone. Therefore, we fabricated a physical model of the left ventricular outflow tract, the mitral valve, the aortic valve and the left ventricle. With this physical model in hand, we were able to visualize the LVOTO and to discuss potential therapeutic options. Moreover, we were able to plan the subsequent redo surgery in detail using the model. This case shows the benefit of 3D printing technologies for surgeons and patients, not only for analysis, but also during the decision-making and pre-operative planning process.

Induced pluripotent stem cells derived from the developing striatum as a potential donor source for cell replacement therapy for Huntington disease.

BACKGROUND: Cell replacement therapy (CRT) for Huntington disease (HD) requires a source of striatal (STR) progenitors capable of restoring the function lost due to STR degeneration. Authentic STR progenitors can be collected from the fetal putative striatum, or whole ganglionic eminence (WGE), but these tissues remain impractical for widespread clinical application, and alternative donor sources are required. Here we begin exploring the possibility that induced pluripotent stem cells (iPSC) derived from WGE may retain an epigenetic memory of their tissue of origin, which could enhance their ability to differentiate into STR cells. RESULTS: We generate four iPSC lines from human WGE (hWGE) and establish that they have a capacity similar to human embryonic stem cells with regard to their ability to differentiate toward an STR phenotype, as measured by expression and demethylation of key STR genes, while maintaining an overall different methylome. Finally, we demonstrate that these STR-differentiated hWGE iPSCs share characteristics with hWGE (i.e., authentic STR tissues) both in vitro and following transplantation into an HD model. Overall, iPSCs derived from human WGE show promise as a donor source for CRT for HD.

Characterization and phase I study of CLR457, an orally bioavailable pan-class I PI3-kinase inhibitor.

Background CLR457 is an orally bioavailable pan-phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor. Methods CLR457 anti-tumor activity and pharmacokinetics (PK) were characterized by in vitro biochemical assays and in vivo tumor xenografts. A first-in-human study was conducted to determine the maximum tolerated dose (MTD), safety, PK, and efficacy of CLR457. Successive cohorts of patients with advanced solid tumors with PI3K pathway activation received increasing CLR457 doses according to a Bayesian escalation model based on the rate of dose limiting toxicity (DLT) in the first 28-day cycle. Results CLR457 inhibited p110α, p110ß, p110δ and p110γ isoforms with an IC50 of 89 ± 29 nM, 56 ± 35 nM, 39 ± 10 nM and 230 ± 31 nM, respectively. CLR457 exhibited dose-dependent antitumor activity and interfered with glucose homeostasis in PI3K-mutant tumor xenografts. 31 patients received doses ranging from 5 to 100 mg. DLTs included grade 3 hyperglycemia and rash (3). In the 100 mg cohort (n = 11), 3 (27.3%) patients had DLTs and all patients (100%) experienced ≥ grade 3 toxicity with rash (45.5%) as the most common event. The MTD was not determined. For the entire study population, stomatitis (45.2%), diarrhea (38.7%), rash (35.5%) were the most common any grade toxicities-51.6% patients experienced ≥ Grade 3 toxicity. CLR457 was rapidly absorbed with limited accumulation and linear PK. PK modeling indicated that pharmacologically active concentrations were achieved at the highest dose tested (100 mg), though no objective responses were observed. Conclusion CLR457 clinical development was terminated due to poor tolerability and limited antitumor activity. These results emphasize the difficulty of achieving a wide therapeutic index when targeting all class I PI3K-isoforms.

A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response.

Microglia are increasingly implicated in brain pathology, particularly neurodegenerative disease, with many genes implicated in Alzheimer's, Parkinson's, and motor neuron disease expressed in microglia. There is, therefore, a need for authentic, efficient in vitro models to study human microglial pathological mechanisms. Microglia originate from the yolk sac as MYB-independent macrophages, migrating into the developing brain to complete differentiation. Here, we recapitulate microglial ontogeny by highly efficient differentiation of embryonic MYB-independent iPSC-derived macrophages then co-culture them with iPSC-derived cortical neurons. Co-cultures retain neuronal maturity and functionality for many weeks. Co-culture microglia express key microglia-specific markers and neurodegenerative disease-relevant genes, develop highly dynamic ramifications, and are phagocytic. Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines. Importantly, co-culture microglia downregulate pathogen-response pathways, upregulate homeostatic function pathways, and promote a more anti-inflammatory and pro-remodeling cytokine response than corresponding monocultures, demonstrating that co-cultures are preferable for modeling authentic microglial physiology.

Limitations of Sulforhodamine 101 for Brain Imaging.

Since 2004, the red fluorescent dye Sulforhodamine 101 (SR101) has been boosting the functional analysis of astrocytes in a functional environment in an unprecedented way. However, two major limitations have been challenging the usefulness of this tool for cellular imaging: (i) SR101 is not as specific for astrocytes as previously reported; and (ii) discoveries of severe excitatory side effects of SR101 are bearing the risk of unwanted alteration of the system of interest. In this article, we summarize the current knowledge about SR101-labeling protocols and discuss the problems that arise from varying of the staining protocols. Furthermore, we provide a testable hypothesis for the observed hyper-excitability that can be observed when using SR101.

Improving and accelerating the differentiation and functional maturation of human stem cell-derived neurons: role of extracellular calcium and GABA.

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABAA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process - from pre-patterned neural progenitor to active neuron - takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia.

Increasing metabolic stability via the deuterium kinetic isotope effect: An example from a proline-amide-urea aminothiazole series of phosphatidylinositol-3 kinase alpha inhibitors.

In vitro metabolic identification studies with a PI3K-α inhibitor lead molecule 1 identified a single predominant site of oxidative metabolism to be occurring within a tert.butyl moiety. Modification of the tert.butyl group within the lead molecule 1, to the corresponding d9-tert.butyl analogue 2, led to an increase in both the in vitro and in vivo metabolic stability. This increase in metabolic stability resulted in a 2-fold increase in the oral bioavailability measured in the rat, and a 3-fold increase in potency in a chronic in vivo study in the mouse, for 2 when compared to 1.

Optimisation of a 5-[3-phenyl-(2-cyclic-ether)-methyl-ether]-4-aminopyrrolopyrimidine series of IGF-1R inhibitors.

Taking the pyrrolopyrimidine derived IGF-1R inhibitor NVP-AEW541 as the starting point, the benzyl ether back-pocket binding moiety was replaced with a series of 2-cyclic ether methyl ethers leading to the identification of novel achiral [2.2.1]-bicyclic ether methyl ether containing analogues with improved IGF-1R activities and kinase selectivities. Further exploration of the series, including a fluorine scan of the 5-phenyl substituent, and optimisation of the sugar-pocket binding moiety identified compound 33 containing (S)-2-tetrahydrofuran methyl ether 6-fluorophenyl ether back-pocket, and cis-N-Ac-Pip sugar-pocket binding groups. Compound 33 showed improved selectivity and pharmacokinetics compared to NVP-AEW541, and produced comparable in vivo efficacy to linsitinib in inhibiting the growth of an IGF-1R dependent tumour xenograft model in the mouse.

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3ß signaling promote functional maturation of induced pluripotent stem cell-derived neurons.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

Norepinephrine-induced calcium signaling in astrocytes in the respiratory network of the ventrolateral medulla.

The neuronal activity in the respiratory network of the ventrolateral medulla strongly depends on a variety of different neuromodulators. Since the respiratory activity generated by neurons in the pre-Bötzinger complex (preBötC) is stabilized by astrocytes, we investigated potential effects of the neuromodulator norepinephrine (NE) on the astrocytic calcium signaling in the ventral respiratory group. In acutely isolated brainstem slices from wild type mice (postnatal day 1-10) we performed calcium imaging experiments using Oregon Green 488 BAPTA-1 AM as a calcium indicator dye. Astrocytes in the preBötC, which were identified by their unique intracellular calcium rise after the reduction of the extracellular K(+) concentration, showed calcium rises in response to norepinephrine. These calcium signals persisted after blockade of neuronal activity by tetrodotoxin (TTX) indicating that they were independent of neuronal activity. Furthermore, application of the endoplasmic reticulum calcium pump blocker cyclopiazonic acid (CPA) diminished norepinephrine-induced calcium signals. This results could be confirmed using transgenic mice with astrocyte specific expression of GCaMP3. Thus, norepinephrine might, apart from acting directly on neurons, influence and modulate respiratory network activity via the modulation of astroglial calcium signaling.

Maximizing the Efficacy of MAPK-Targeted Treatment in PTENLOF/BRAFMUT Melanoma through PI3K and IGF1R Inhibition.

The introduction of MAPK pathway inhibitors paved the road for significant advancements in the treatment of BRAF-mutant (BRAF(MUT)) melanoma. However, even BRAF/MEK inhibitor combination therapy has failed to offer a curative treatment option, most likely because these pathways constitute a codependent signaling network. Concomitant PTEN loss of function (PTEN(LOF)) occurs in approximately 40% of BRAF(MUT) melanomas. In this study, we sought to identify the nodes of the PTEN/PI3K pathway that would be amenable to combined therapy with MAPK pathway inhibitors for the treatment of PTEN(LOF)/BRAF(MUT) melanoma. Large-scale compound sensitivity profiling revealed that PTEN(LOF) melanoma cell lines were sensitive to PI3Kß inhibitors, albeit only partially. An unbiased shRNA screen (7,500 genes and 20 shRNAs/genes) across 11 cell lines in the presence of a PI3Kß inhibitor identified an adaptive response involving the IGF1R-PI3Kα axis. Combined inhibition of the MAPK pathway, PI3Kß, and PI3Kα or insulin-like growth factor receptor 1 (IGF1R) synergistically sustained pathway blockade, induced apoptosis, and inhibited tumor growth in PTEN(LOF)/BRAF(MUT) melanoma models. Notably, combined treatment with the IGF1R inhibitor, but not the PI3Kα inhibitor, failed to elevate glucose or insulin signaling. Taken together, our findings provide a strong rationale for testing combinations of panPI3K, PI3Kß + IGF1R, and MAPK pathway inhibitors in PTEN(LOF)/BRAF(MUT) melanoma patients to achieve maximal response.

A Novel Potent Oral Series of VEGFR2 Inhibitors Abrogate Tumor Growth by Inhibiting Angiogenesis.

This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays, the compounds exhibit single-digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the tumor vasculature as demonstrated by ELISA for TIE-2 in lysates or by immunohistochemical analysis. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.