Guillermo A. Morales

Dual-activity PI3K-BRD4 inhibitor for the orthogonal inhibition of MYC to block tumor growth and metastasis.

Significance In this work, we describe a dual-action inhibitor that simultaneously disrupts functions of two key MYC-mediating factors—PI3K and BRD4. We show that the concomitant inhibition of PI3K and BRD4 blocks MYC expression and activation, promotes MYC degradation, and markedly inhibits cancer cell growth and metastasis. Our findings suggest that the dual-activity inhibitor represents a highly promising lead compound for the development of novel anticancer therapeutics. MYC is a major cancer driver but is documented to be a difficult therapeutic target itself. Here, we report on the biological activity, the structural basis, and therapeutic effects of the family of multitargeted compounds that simultaneously disrupt functions of two critical MYC-mediating factors through inhibiting the acetyllysine binding of BRD4 and the kinase activity of PI3K. We show that the dual-action inhibitor impairs PI3K/BRD4 signaling in vitro and in vivo and affords maximal MYC down-regulation. The concomitant inhibition of PI3K and BRD4 blocks MYC expression and activation, promotes MYC degradation, and markedly inhibits cancer cell growth and metastasis. Collectively, our findings suggest that the dual-activity inhibitor represents a highly promising lead compound for the development of novel anticancer therapeutics.

Synthesis and cancer stem cell-based activity of substituted 5-morpholino-7H-thieno[3,2-b]pyran-7-ones designed as next generation PI3K inhibitors.

Dysregulation of the phosphatidylinositol-3-kinase (PI3K) pathway in a wide range of tumors has made PI3K a consensus target to inhibit as illustrated by more than 15 inhibitors now in clinical trials. Our previous work, built on the early pioneering multikinase inhibitor LY294002, resulted in the only PI3K vascular-targeted PI3K inhibitor prodrug, SF1126, which has now completed Phase I clinical trials. This inhibitor has properties that impart more in vivo activity than should be warranted by its enzymatic potency, which in general is much lower than other clinical stage PI3K inhibitors. We embarked on the exploration of scaffolds that retained such properties while simultaneously exhibiting an increased potency toward PI3K. This work resulted in the discovery of the 5-morpholino-7H-thieno[3,2-b]pyran-7-one system as the foundation of a new compound class of potential PI3K inhibitors having improved potency toward PI3K. The synthesis and cancer stem cell-based activity of these compounds are reported herein.

PI-3K Inhibitors Preferentially Target CD15+ Cancer Stem Cell Population in SHH Driven Medulloblastoma

Sonic hedgehog (SHH) medulloblastoma (MB) subtype is driven by a proliferative CD15+ tumor propagating cell (TPC), also considered in the literature as a putative cancer stem cell (CSC). Despite considerable research, much of the biology of this TPC remains unknown. We report evidence that phosphatase and tensin homolog (PTEN) and phosphoinositide 3-kinase (PI-3K) play a crucial role in the propagation, survival and potential response to therapy in this CD15+ CSC/TPC-driven malignant disease. Using the ND2-SmoA1 transgenic mouse model for MB, mouse genetics and patient-derived xenografts (PDXs), we demonstrate that the CD15+TPCs are 1) obligately required for SmoA1Tg-driven tumorigenicity 2) regulated by PTEN and PI-3K signaling 3) selectively sensitive to the cytotoxic effects of pan PI-3K inhibitors in vitro and in vivo but resistant to chemotherapy 4) in the SmoA1Tg mouse model are genomically similar to the SHH human MB subgroup. The results provide the first evidence that PTEN plays a role in MB TPC signaling and biology and that PI-3K inhibitors target and suppress the survival and proliferation of cells within the mouse and human CD15+ cancer stem cell compartment. In contrast, CD15+ TPCs are resistant to cisplatinum, temozolomide and the SHH inhibitor, NVP-LDE-225, agents currently used in treatment of medulloblastoma. These studies validate the therapeutic efficacy of pan PI-3K inhibitors in the treatment of CD15+ TPC dependent medulloblastoma and suggest a sequential combination of PI-3K inhibitors and chemotherapy will have augmented efficacy in the treatment of this disease.

Single Agent and Synergistic Activity of the “First-in-Class” Dual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma

Deregulated PI3K/AKT/mTOR, Ras/Raf/MAPK, and c-Myc signaling pathways are of prognostic significance in hepatocellular carcinoma (HCC). Sorafenib, the only drug clinically approved for patients with advanced HCC, blocks the Ras/Raf/MAPK pathway but it does not inhibit the PI3K/AKT/mTOR pathway or c-Myc activation. Hence, there is an unmet medical need to identify potent PI3K/BRD4 inhibitors, which can be used either alone or in combination with sorafenib to treat patients with advanced HCC. Herein, we show that SF1126 (pan PI3K/BRD4 inhibitor) as single agent or in combination with sorafenib inhibited proliferation, cell cycle, apoptosis, and multiple key enzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK pathway in Hep3B, HepG2, SK-Hep1, and Huh7 HCC cell lines. We demonstrate that the active moiety of the SF1126 prodrug LY294002 binds to and blocks BRD4 interaction with the acetylated histone-H4 chromatin mark protein and displaced BRD4 coactivator protein from the transcriptional start site of MYC in Huh7 and SK-Hep1 HCC cell lines. Moreover, SF1126 blocked expression levels of c-Myc in HCC cells. Treatment of SF1126 either alone or in combination with sorafenib showed significant antitumor activity in vivo. Our results establish that SF1126 is a dual PI3K/BRD4 inhibitor. This agent has completed a phase I clinical trial in humans with good safety profile. Our data support the potential future consideration of a phase II clinical trial of SF1126, a clinically relevant dual “first-in-class” PI3K/BRD4 inhibitor in advanced HCC, and a potential combination with sorafenib. Mol Cancer Ther; 15(11); 2553–62. ©2016 AACR.

Association of high microvessel α v β 3 and low PTEN with poor outcome in stage 3 neuroblastoma: rationale for using first in class dual PI3K/BRD4 inhibitor, SF1126

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvβ3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvβ3 was expressed on 68% of microvessels in MYCN-amplified stage 3 neuroblastomas, but only on 34% (means) in MYCN-non-amplified tumors (p < 0.001; n = 54). PTEN, a tumor suppressor involved in αvβ3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvβ3 was expressed on 60% of tumor microvessels when PTEN was negative or focal, as compared to 32% of microvessels in tumors with diffuse PTEN expression (p < 0.001). In a MYCN transgenic mouse model, loss of one allele of PTEN promoted tumor growth, illustrating the potential role of PTEN in neuroblastoma pathogenesis. Interestingly, we report the novel dual PI-3K/BRD4 activity of SF1126 (originally developed as an RGD-conjugated pan PI3K inhibitor). SF1126 inhibits BRD4 bromodomain binding to acetylated lysine residues with histone H3 as well as PI3K activity in the MYCN amplified neuroblastoma cell line IMR-32. Moreover, SF1126 suppressed MYCN expression and MYCN associated transcriptional activity in IMR-32 and CHLA136, resulting in overall decrease in neuroblastoma cell viability. Finally, treatment of neuroblastoma tumors with SF1126 inhibited neuroblastoma growth in vivo. These data suggest integrin αvβ3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvβ3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma.Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvβ3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvβ3 was expressed on 68% of microvessels in MYCN-amplified stage 3 neuroblastomas, but only on 34% (means) in MYCN-non-amplified tumors (p < 0.001; n = 54). PTEN, a tumor suppressor involved in αvβ3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvβ3 was expressed on 60% of tumor microvessels when PTEN was negative or focal, as compared to 32% of microvessels in tumors with diffuse PTEN expression (p < 0.001). In a MYCN transgenic mouse model, loss of one allele of PTEN promoted tumor growth, illustrating the potential role of PTEN in neuroblastoma pathogenesis. Interestingly, we report the novel dual PI-3K/BRD4 activity of SF1126 (originally developed as an RGD-conjugated pan PI3K inhibitor). SF1126 inhibits BRD4 bromodomain binding to acetylated lysine residues with histone H3 as well as PI3K activity in the MYCN amplified neuroblastoma cell line IMR-32. Moreover, SF1126 suppressed MYCN expression and MYCN associated transcriptional activity in IMR-32 and CHLA136, resulting in overall decrease in neuroblastoma cell viability. Finally, treatment of neuroblastoma tumors with SF1126 inhibited neuroblastoma growth in vivo. These data suggest integrin αvβ3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvβ3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma.

Abstract LB-211: A novel dual epigenetic/kinase inhibitory platform: SF2523 and analogues as first-in-class PI3K/BRD4 inhibitors

The transcription factor MYC (c-MYC and MYCN) plays a key role in cancer but small molecule inhibitors of it have been elusive. A new series of PI3K inhibitors has been under investigation and through molecular modeling compounds have been designed to also inhibit the bromodomain protein BRD4. We used structure-based drug design to create a series of anticancer compounds based on a thieno-pyranone core that potently inhibit both the epigenetic regulator bromodomain protein BRD4 and the key nodal survival signaling kinase PI3K. Two lead dual PI3K/BRD4 inhibitors from our research are: pan-PI3K inhibitor SF2523 (PI3K alpha, beta, delta, gamma IC50 values in nM: 17, 214, 27, 232) inhibiting BRD4-1 and BRD4-2 (IC50 = 318 nM and 1660 nM), and delta-PI3K selective inhibitor SF2535 (PI3K alpha, beta, delta, gamma IC50 values in nM: 714, 1750, 27, 1170) inhibiting BRD4-1 and BRD4-2 (IC50 = 277 nM and 628 nM). Our results indicate that SF2523 blocks BRD4 binding to MYCN promoter PS1/PS2 using chromatin precipitation experiments. This approach provides for the first time access to inhibit the activity of MYC by enhancing its degradation (PI3K inhibition) and blocking MYC production via the inhibition of MYC transcription (BRD4 inhibition). Our studies demonstrate that SF2523 blocks M1-M2 transition and decreases levels of p-AKT, N-MYC in several neuroblastoma cell lines as well as in vivo simultaneous pharmacodynamic knockdown. Other in vivo studies show SF2523 blocks spontaneous metastasis and tumor growth. To date, SF2523 has demonstrated animal efficacy results without toxicity in the following 4 animal models: orthotopic pancreatic model, multiple myeloma model, renal cell carcinoma model, neuroblastoma xenograft model. The crystal structure of the dual PI3K/BRD4 inhibitor SF2523 with BRD4 has been determined providing key insights towards the design of more potent analogs. Our studies provide evidence that inhibition of PI3K-gamma and PI3K-delta by our thieno-pyranone chemotypes function as checkpoint inhibitors and enhance immune-therapeutics while BRD4 inhibition has been shown to block tumor-specific super-enhancers activating the innate and adaptive immune response providing a novel strategy to treat cancer. These dual inhibitors represent a new therapeutic approach inhibiting two key anticancer orthogonal mechanisms, BRD4 and PI3K, showing significant efficacy justifying further development efforts towards clinical evaluation. Citation Format: Alok Singh, Shweta Joshi, Joseph R. Garlich, Guillermo Morales, Tatiana Kutateladze, Forest Andrews, Donald Durden. A novel dual epigenetic/kinase inhibitory platform: SF2523 and analogues as first-in-class PI3K/BRD4 inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-211.

Abstract LB-298: The novel triple PI3K-CDK4/6-BRD4 inhibitor SRX3177 harnesses synthetic lethality relationships to orthogonally disrupt cancer cell signaling

Dysregulation of the cell cycle is a hallmark of nearly all cancers, and efforts to target signaling pathways regulating cell growth and proliferation have driven much of cancer drug discovery. Despite advances in novel therapeutics, however, most patients with advanced neoplasms do not achieve long-term survival with single agent targeted therapy. Here, we describe a novel “triple inhibitor” (i.e., SRX3177) that simultaneously targets three oncogenes promoting cancer cell growth: phosphatidylinositol-3 kinase (PI3K), cyclin-dependent kinases 4 and 6 (CDK4/6), and the epigenetic regulator BRD4. This rationally-designed, thieno-pyranone scaffold-based small molecule inhibitor uses known synthetic lethality relationships to orthogonally disrupt three targets within the cancer cell with one agent. Single agent CDK4/6 inhibitors such as palbociclib, which is FDA-approved in combination with hormone therapy in estrogen-receptor positive breast cancer, suffer from being cytostatic in nature, requiring combinations to be more effective and avoid development of resistance. Concurrent PI3K inhibition can prevent resistance to CDK4/6 inhibition, and combined CDK4/6 and PI3K inhibition leads to synthetic lethality reported in a number of cancer types, including breast cancer and mantle cell lymphoma. Moreover, blocking the chromatin reader protein BRD4 downregulates MYC and cyclin D1 transcription, further promoting cell cycle arrest in G1. Thus, we designed SRX3177 as a triple inhibitor of PI3K, CDK4/6, and BRD4 to maximally block cell cycle progression and cancer cell growth. SRX3177 is a potent ATP competitive CDK4/6 inhibitor (IC50: CDK4 = 2.54 nM, CDK6 = 3.26 nM), PI3K inhibitor (IC50: PI3Kα = 79.3 nM, PI3Kδ = 83.4 nM), and BRD4 inhibitor (IC50: BD1 = 32.9 nM, BD2 = 88.8 nM). In a panel of mantle cell lymphoma, neuroblastoma, and hepatocellular carcinoma cell lines, SRX3177 has maximal IC50 values of 578 nM, 385 nM, and 495 nM respectively. This represents a 19 to 82-fold increase in potency compared to palbociclib. SRX3177 is 5-fold more potent in cancer cells than the combination of similar potency single PI3K, CD4/6, and BRD4 inhibitors (i.e., BKM120 + palbociclib + JQ1). SRX3177 is also 40-fold less toxic to normal epithelial cells than the co-treatment with single inhibitors. Furthermore, SRX3177 induces cell cycle arrest and apoptosis in propidium iodide and annexin V assays, respectively. Finally, SRX3177 inhibits Akt and Rb phosphorylation (downstream of PI3K and CDK4/6 signaling, respectively) and blocks BRD4 binding to chromatin. Thus, our triple inhibitor SRX3177 is efficacious, is more potent and less toxic to normal cells than administration of three individual inhibitors, and has robust pharmacodynamic effects on its targets. Taken together, our data support the development of SRX3177 as a novel therapeutic agent for multiple cancers. Citation Format: Adam M. Burgoyne, Francisco M. Vega, Alok Singh, Shweta Joshi, Joseph R. Garlich, Guillermo A. Morales, Tatiana G. Kutateladze, Donald L. Durden. The novel triple PI3K-CDK4/6-BRD4 inhibitor SRX3177 harnesses synthetic lethality relationships to orthogonally disrupt cancer cell signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-298. doi:10.1158/1538-7445.AM2017-LB-298

Abstract A27: Dual PI3K/BRD4 (kinase/epigenetic) inhibitors for maximal MYC control in cancer therapeutics

There is an unmet need to inhibit the key cancer promoting transcription factor MYC. MYC (both cMYC and MYCN) acts downstream of many cell receptor complexes and signal transduction pathways to activate genes that drive cancer cell growth and proliferation. To date, small molecule inhibitors of MYC have been elusive. It has recently been shown by others that PI3K inhibitors can help degrade the MYC protein. It has also been shown by others that inhibitors of the bromodomain protein BRD4 serve to block the transcription of MYC. Our innovative approach is to indirectly orthogonally diminish the activity of MYC by enhancing its degradation using PI3 kinase (PI3K) inhibitors combined with blocking transcription of the gene producing MYC using inhibitors of the bromodomain protein BRD4. While small molecule inhibitors or PI3K and BRD4 are individually used in cancer clinical trials, none has made it through development to FDA approval. Although combination treatments are common in cancer, there is an unmet need for ever increasing combinations to inhibit multiple targets to maximize efficacy. This becomes unfeasible due to prohibitive costs when combining expensive targeted therapies in addition to being a barrier to early clinical evaluation of such complex combinations of drugs. While exploring a novel PI-3K inhibitor scaffold (TP Scaffold) based on a chromone derivative we discovered that these compounds also can potently inhibit BRD4. The chromone backbone is common to the inhibition of BOTH targets thus providing the opportunity to now explore the BRD4 inhibitory activity while maintaining potent PI3K activity. Consolidating the inhibition of PI3K AND BRD4 to maximally inhibit MYC activity with one molecule would fill all the unmet needs stated above. We have identified a lead compound, SF2523 that inhibits BRD4 (BRD4-1 IC50 241 nM, BRD4-2 IC50 1550 nM) and also is a pan-PI3K inhibitor (alpha, beta, delta, gamma IC50 34, 214, 960, and 158 nM respectively). We have demonstrated suitable ADME and PK properties for SF2523. The following preclinical results will be presented: a) Western blots demonstrating dual pathway inhibition; b) in vitro studies proving decreased MYC levels; c) antiproliferation data including patient derived cancer cells; d) antiangiogenic activity; e) induction of apoptosis and mechanisms thereof. Additionally, we will present data showing safety and efficacy of the dual PI3K/BRD4 inhibitor SF2523 in animal efficacy models including an orthotopic pancreatic model and Lewis Lung Carcinoma mouse model. In addition to the PI3K/BRD4 inhibitor our recent results with designed dual inhibitors within a single molecule aimed at PI3K/MEK, PI3K/HDAC, and other critical combinations will be presented. Our approach challenges the current dogma in the PI3K inhibitor field because it attacks a key cancer target using two orthogonal mechanisms within a single compound as well as challenges the cost-ignoring efficacy-limiting mentality of one-drug one-target prevalent in cancer research in an area with no FDA approved treatments. The results highlight the development of one of the first nanomolar potent dual PI-3K/BET bromodomain inhibitors for clinical development against PI-3K and epigenetic targets in the cancer cell. Citation Format: Alok R. Singh, Shweta Joshi, Joseph R. Garlich, Guillermo A. Morales, Donald Lee Durden. Dual PI3K/BRD4 (kinase/epigenetic) inhibitors for maximal MYC control in cancer therapeutics. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A27.