CAL-101, a Potent Selective Inhibitor of the p110d Isoform of Phosphatidylinositol 3-Kinase, Attenuates PI3K Signaling and Inhibits Proliferation and Survival of Acute Lympoblastic Leukemia in Addition to a Range of Other Hematological Malignancies
Authors: Lannutti Brian J , Meadows Sarah A. , Kashishian Adam , Steiner Bart , May Sarah , Johnson Amy J. , Ulrich Roger G. , Yu Albert , Gallatin Michael W. , Byrd John C. , Puri Kamal D. , Giese Neill A. 
Published: April 2020

Abstract
The class I phosphatidylinositol 3-kinases (PI3K) regulate a variety of cellular functions including motility, metabolism, proliferation, growth, and survival, depending on cellular milieu. Deregulation of the PI3K/Akt pathway is one of the most frequently observed defects in human malignancies including those of hematological origin and has been shown to play an important role in tumor progression. Therefore, selective targeting of PI3K signaling in hematological tumor cells could provide an effective treatment strategy while limiting potential undesirable effects of pan-inhibitors that broadly block PI3K signaling in all cells. Of the class IA PI3Ks (p110a, p110b, p110d), p110d’s expression is largely restricted to cells of hematopoietic origin and is essential for PI3K signaling in lymphocytes. Here, we report on the characterization of a novel p110d specific inhibitor, CAL-101. This compound is a potent PI3K inhibitor with an IC50 of 1–10 nM against the purified p110d subunit and 30–70 nM cellular potency against p110d-mediated basophil activation in whole blood. Importantly, CAL-101 plasma concentrations of 500–5000 nM that greatly exceed those needed for p110d inhibition in blood were safely maintained in a 7 day multidose normal human volunteer study. CAL-101 demonstrates &gt;30-fold selectivity over other class I, II and III PI3K family members as well as selectivity over other PI3K-related proteins including mTOR and DNA-PK. Furthermore, a genome wide screen of &gt;350 protein kinases did not detect any activity. To investigate the potential role of p110d in hematologic tumors we screened a wide range of leukemia and lymphoma cell lines for p110 isoform expression and constitutive PI3K pathway activation. The expression of p110d was observed in &gt;90% of these cell lines that was in many cases accompanied by constitutive Akt phosphorylation. In this context, CAL-101 was able to reduce p-Akt levels and block additional downstream effectors such as p-p70S6K, p-GSKb, and p-Bad in cells that represent a range of tumor types including acute myeloid leukemia, acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma among others. Recent studies have demonstrated the importance of PTEN loss and enhanced PI3K signaling in primary T-ALL cells. We report high levels of p110d protein and activated Akt in 6 of 6 ALL cell lines evaluated. Inhibition of p110d with CAL-101 treatment of both T-ALL and B-ALL cell lines resulted in a reduction of Akt and GSK-3b phosphorylation and a decrease in cellular proliferation that was accompanied by cell death demonstrating an essential role of PI3K signaling independent of PTEN status. Treatment of T-ALL cell lines with CAL-101 induced processing of pro-caspase-3 and cleavage of PARP supporting a role for caspase mediated cell death. These studies have now been extended to the analysis of primary patient blast samples to further establish preclinical proof of concept for therapeutic application of CAL-101 for the treatment of ALL. In summary, CAL-101 is a highly potent and selective p110d kinase inhibiter with broad anti-tumor activity against cancer cells of hematologic origin. Clinical studies in normal human volunteers demonstrated good tolerability with high drug exposure and favorable steady-state pharmacokinetic properties. Taken together, these data support the on going Phase 1 clinical trial that includes a wide range of hematological malignancies.