A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Intermittent PI3Kδ inhibition sustains anti-tumour immunity and curbs irAEs.

Phosphoinositide 3-kinase δ (PI3Kδ) has a key role in lymphocytes, and inhibitors that target this PI3K have been approved for treatment of B cell malignancies1-3. Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity4,5, its effect on solid tumours in humans remains unclear. Here we assessed the effects of the PI3Kδi AMG319 in human patients with head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomized phase II trial (EudraCT no. 2014-004388-20). PI3Kδ inhibition decreased the number of tumour-infiltrating regulatory T (Treg) cells and enhanced the cytotoxic potential of tumour-infiltrating T cells. At the tested doses of AMG319, immune-related adverse events (irAEs) required treatment to be discontinued in 12 out of 21 of patients treated with AMG319, suggestive of systemic effects on Treg cells. Accordingly, in mouse models, PI3Kδi decreased the number of Treg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 Treg cells, accompanied by expansion of pathogenic T helper 17 (TH17) and type 17 CD8+ T (TC17) cells, which probably contributed to toxicity; this points towards a specific mode of action for the emergence of irAEs. A modified treatment regimen with intermittent dosing of PI3Kδi in mouse models led to a significant decrease in tumour growth without inducing pathogenic T cells in colonic tissue, indicating that alternative dosing regimens might limit toxicity.

Enhanced antitumor immunity through sequential targeting of PI3Kδ and LAG3.

BACKGROUND: Despite striking successes, immunotherapies aimed at increasing cancer-specific T cell responses are unsuccessful in most patients with cancer. Inactivating regulatory T cells (Treg) by inhibiting the PI3Kδ signaling enzyme has shown promise in preclinical models of tumor immunity and is currently being tested in early phase clinical trials in solid tumors. METHODS: Mice bearing 4T1 mammary tumors were orally administered a PI3Kδ inhibitor (PI-3065) daily and tumor growth, survival and T cell infiltrate were analyzed in the tumor microenvironment. A second treatment schedule comprised PI3Kδ inhibitor with anti-LAG3 antibodies administered sequentially 10 days later. RESULTS: As observed in human immunotherapy trials with other agents, immunomodulation by PI3Kδ-blockade led to 4T1 tumor regressor and non-regressor mice. Tumor infiltrating T cells in regressors were metabolically fitter than those in non-regressors, with significant enrichments of antigen-specific CD8+ T cells, T cell factor 1 (TCF1)+ T cells and CD69- T cells, compatible with induction of a sustained tumor-specific T cell response. Treg numbers were significantly reduced in both regressor and non-regressor tumors compared with untreated tumors. The remaining Treg in non-regressor tumors were however significantly enriched with cells expressing the coinhibitory receptor LAG3, compared with Treg in regressor and untreated tumors. This striking difference prompted us to sequentially block PI3Kδ and LAG3. This combination enabled successful therapy of all mice, demonstrating the functional importance of LAG3 in non-regression of tumors on PI3Kδ inhibition therapy. Follow-up studies, performed using additional cancer cell lines, namely MC38 and CT26, indicated that a partial initial response to PI3Kδ inhibition is an essential prerequisite to a sequential therapeutic benefit of anti-LAG3 antibodies. CONCLUSIONS: These data indicate that LAG3 is a key bottleneck to successful PI3Kδ-targeted immunotherapy and provide a rationale for combining PI3Kδ/LAG3 blockade in future clinical studies.

TGFß inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.

Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy. However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction of paracrine senescence was observed within 24 hours and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence, we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended on macrophage-derived transforming growth factor-ß1 (TGFß1) ligand. In acetaminophen poisoning, inhibition of TGFß receptor 1 (TGFßR1) improved mouse survival. TGFßR1 inhibition reduced senescence and enhanced liver regeneration even when delivered beyond the therapeutic window for treating acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

PI3Kα inhibition reduces obesity in mice.

Partial inhibition of PI3K is one of the best-validated and evolutionary conserved manipulations to extend longevity. The best known health beneficial effects of reduced PI3K are related to metabolism and include increased energy expenditure, reduced nutrient storage, and protection from obesity. We have previously shown that a dual chemical inhibitor of the alpha and delta PI3K isoforms (CNIO-PI3Ki) reduces obesity in mice and monkeys, without evident toxic effects after long-term treatment. Here, we dissect the role of the alpha and delta PI3K isoforms by making use of selective inhibitors against PI3Kα (BYL-719 also known as alpelisib) or PI3Kδ (GS-9820 also known as acalisib). Treatment of mice with the above mentioned inhibitors indicated that BYL-719 increases energy expenditure in normal mice and efficiently reduces body weight in obese (ob/ob) mice, whereas these effects were not observed with GS-9820. Of note, the dose of BYL-719 required to reduce obesity was 10x higher than the equivalent dose of CNIO-PI3Ki, which could suggest that simultaneous inhibition of PI3K alpha and delta is more beneficial than single inhibition of the alpha isoform. In summary, we conclude that inhibition of PI3Kα is sufficient to increase energy expenditure and reduce obesity, and suggest that concomitant PI3Kα inhibition could play an auxiliary role.

p21Cip1 plays a critical role in the physiological adaptation to fasting through activation of PPARα.

Fasting is a physiological stress that elicits well-known metabolic adaptations, however, little is known about the role of stress-responsive tumor suppressors in fasting. Here, we have examined the expression of several tumor suppressors upon fasting in mice. Interestingly, p21 mRNA is uniquely induced in all the tissues tested, particularly in liver and muscle (>10 fold), and this upregulation is independent of p53. Remarkably, in contrast to wild-type mice, p21-null mice become severely morbid after prolonged fasting. The defective adaptation to fasting of p21-null mice is associated to elevated energy expenditure, accelerated depletion of fat stores, and premature activation of protein catabolism in the muscle. Analysis of the liver transcriptome and cell-based assays revealed that the absence of p21 partially impairs the transcriptional program of PPARα, a key regulator of fasting metabolism. Finally, treatment of p21-null mice with a PPARα agonist substantially protects them from their accelerated loss of fat upon fasting. We conclude that p21 plays a relevant role in fasting adaptation through the positive regulation of PPARα.

Pharmacological inhibition of PI3K reduces adiposity and metabolic syndrome in obese mice and rhesus monkeys.

Genetic inhibition of PI3K signaling increases energy expenditure, protects from obesity and metabolic syndrome, and extends longevity. Here, we show that two pharmacological inhibitors of PI3K, CNIO-PI3Ki and GDC-0941, decrease the adiposity of obese mice without affecting their lean mass. Long-term treatment of obese mice with low doses of CNIO-PI3Ki reduces body weight until reaching a balance that is stable for months as long as the treatment continues. CNIO-PI3Ki treatment also ameliorates liver steatosis and decreases glucose serum levels. The above observations have been recapitulated in independent laboratories and using different oral formulations of CNIO-PI3Ki. Finally, daily oral treatment of obese rhesus monkeys for 3 months with low doses of CNIO-PI3Ki decreased their adiposity and lowered their serum glucose levels, in the absence of detectable toxicities. Therefore, pharmacological inhibition of PI3K is an effective and safe anti-obesity intervention that could reverse the negative effects of metabolic syndrome in humans.

Pten positively regulates brown adipose function, energy expenditure, and longevity.

Aging in worms and flies is regulated by the PI3K/Akt/Foxo pathway. Here we extend this paradigm to mammals. Pten(tg) mice carrying additional genomic copies of Pten are protected from cancer and present a significant extension of life span that is independent of their lower cancer incidence. Interestingly, Pten(tg) mice have an increased energy expenditure and protection from metabolic pathologies. The brown adipose tissue (BAT) of Pten(tg) mice is hyperactive and presents high levels of the uncoupling protein Ucp1, which we show is a target of Foxo1. Importantly, a synthetic PI3K inhibitor also increases energy expenditure and hyperactivates the BAT in mice. These effects can be recapitulated in isolated brown adipocytes and, moreover, implants of Pten(tg) fibroblasts programmed with Prdm16 and Cebpß form subcutaneous brown adipose pads more efficiently than wild-type fibroblasts. These observations uncover a role of Pten in promoting energy expenditure, thus decreasing nutrient storage and its associated damage.