Search

Supplementary Table from A First-in-Class Inhibitor of ER Coregulator PELP1 Targets ER+ Breast Cancer

Supplementary Data from A First-in-Class Inhibitor of ER Coregulator PELP1 Targets ER+ Breast Cancer

Supplementary Table 1

Purpose: Salt-inducible kinase 2 (SIK2) is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here, we examine the effects of a novel small-molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer.Experimental Design: SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear–centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death, and reduce AKT/survivin signaling.Results: SIK2 is overexpressed in approximately 30% of high-grade serous ovarian cancers. ARN-3236 inhibited the growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 μmol/L, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson r = −0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip (P = 0.028) and OVCAR8 xenografts. In at least three cell lines, a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest, and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression.Conclusions: ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity. Clin Cancer Res; 23(8); 1945–54. ©2016 AACR.

Supplementary Figure 1 related to Figure 2. (A) In vitro kinetic curve of SIK2 kinase activity in the presence of ARN-3236. The reaction mixture contained SIK2 recombinant protein (15 nM), ATP (10 μM), and substrate peptide (TORC2 aa162-179) (1 μM). (B-J) Indicated cells were treated same as in Figure 1B, Cell viability was tested Supplementary Figure 2 related to Figure 3. (A-H) Different cell lines were plated at density of 8000/well in 96-well plate, then sequentially treated with ARN-3236 (IC30 of each cell line) or DMSO for 24 hr and with increasing concentration of paclitaxel for another 72 hr. Cell viability was measured with an SRB assay. Supplementary Figure 3 related to Figure 4. (A) A2780 cells were transfected with the indicated siRNA or treated with DMSO or ARN-3236 (1 μM). The average nuclear centrosome distance and the percentage of cells with NCU was calculated and plotted. (B, C) OVCAR8 and A2780 cells were treated as in Figure 4B. Bar plots represent the mean {plus minus} s.d. of the percentage of cells at different stage of mitosis (left plot) and the mean prometaphase/metaphase ratio {plus minus} s.d (right plot). Supplementary Figure 4 related to Figure 5. (A) OC316 and OVCAR8 ovarian cancer cells were treated as in Figure 5A, fixed, stained with PI, and subjected to cell cycle analysis. (B) OC316 and OVCAR8 ovarian cancer cells were treated as in Figure 5B, the representative percentage of apoptotic cells is shown. Supplementary Figure 5 related to Figure 6. (A) The expression of 272 proteins was measured using a reverse phase protein array after treatment of SKOv3 ovarian cancer cells with 2 μM of ARN-3236 for 48 hrs. The log2 (fold change) of each protein were plotted. (B) OC316, A2780 and OVCAR8 ovarian cancer cell lines were treated with ARN-3236 for 48 hrs, cell lysates were collected and analyzed by western blot for AKT-pS473, AKT-pT308, AKT and survivn, GAPDH was used as a loading control. (C) Western analysis of phosphorylation of AKT and survivin was performed using three vehicle and ARN-3236 treated tumor cell lysates.

Supplementary Figure from A First-in-Class Inhibitor of ER Coregulator PELP1 Targets ER+ Breast Cancer

Most patients with estrogen receptor alpha–positive (ER+) breast cancers initially respond to treatment but eventually develop therapy resistance with disease progression. Overexpression of oncogenic ER coregulators, including proline, glutamic acid, and leucine-rich protein 1 (PELP1), are implicated in breast cancer progression. The lack of small molecules that inhibits PELP1 represents a major knowledge gap. Here, using a yeast-two-hybrid screen, we identified novel peptide inhibitors of PELP1 (PIP). Biochemical assays demonstrated that one of these peptides, PIP1, directly interacted with PELP1 to block PELP1 oncogenic functions. Computational modeling of PIP1 revealed key residues contributing to its activity and facilitated the development of a small-molecule inhibitor of PELP1, SMIP34, and further analyses confirmed that SMIP34 directly bound to PELP1. In breast cancer cells, SMIP34 reduced cell growth in a dose-dependent manner. SMIP34 inhibited proliferation of not only wild-type (WT) but also mutant (MT) ER+ and therapy-resistant breast cancer cells, in part by inducing PELP1 degradation via the proteasome pathway. RNA sequencing analyses showed that SMIP34 treatment altered the expression of genes associated with estrogen response, cell cycle, and apoptosis pathways. In cell line–derived and patient-derived xenografts of both WT and MT ER+ breast cancer models, SMIP34 reduced proliferation and significantly suppressed tumor progression. Collectively, these results demonstrate SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling in advanced breast cancer.Significance:Development of a novel inhibitor of oncogenic PELP1 provides potential therapeutic avenues for treating therapy-resistant, advanced ER+ breast cancer.

Most patients with estrogen receptor alpha–positive (ER+) breast cancers initially respond to treatment but eventually develop therapy resistance with disease progression. Overexpression of oncogenic ER coregulators, including proline, glutamic acid, and leucine-rich protein 1 (PELP1), are implicated in breast cancer progression. The lack of small molecules that inhibits PELP1 represents a major knowledge gap. Here, using a yeast-two-hybrid screen, we identified novel peptide inhibitors of PELP1 (PIP). Biochemical assays demonstrated that one of these peptides, PIP1, directly interacted with PELP1 to block PELP1 oncogenic functions. Computational modeling of PIP1 revealed key residues contributing to its activity and facilitated the development of a small-molecule inhibitor of PELP1, SMIP34, and further analyses confirmed that SMIP34 directly bound to PELP1. In breast cancer cells, SMIP34 reduced cell growth in a dose-dependent manner. SMIP34 inhibited proliferation of not only wild-type (WT) but also mutant (MT) ER+ and therapy-resistant breast cancer cells, in part by inducing PELP1 degradation via the proteasome pathway. RNA sequencing analyses showed that SMIP34 treatment altered the expression of genes associated with estrogen response, cell cycle, and apoptosis pathways. In cell line–derived and patient-derived xenografts of both WT and MT ER+ breast cancer models, SMIP34 reduced proliferation and significantly suppressed tumor progression. Collectively, these results demonstrate SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling in advanced breast cancer. Significance: Development of a novel inhibitor of oncogenic PELP1 provides potential therapeutic avenues for treating therapy-resistant, advanced ER+ breast cancer.

The central role of β-catenin in the Wnt pathway makes it an attractive therapeutic target for cancers driven by aberrant Wnt signaling. We recently developed a small-molecule inhibitor, BC-2059, that promotes apoptosis by disrupting the β-catenin/transducin β-like 1 (TBL1) complex through an unknown mechanism of action. In this study, we show that BC-2059 directly interacts with high affinity for TBL1 when in complex with β-catenin. We identified two amino acids in a hydrophobic pocket of TBL1 that are required for binding with β-catenin, and computational modeling predicted that BC-2059 interacts at the same hydrophobic pocket. Although this pocket in TBL1 is involved in binding with NCoR/SMRT complex members G Protein Pathway Suppressor 2 (GSP2) and SMRT and p65 NFκB subunit, BC-2059 failed to disrupt the interaction of TBL1 with either NCoR/SMRT or NFκB. Together, our results show that BC-2059 selectively targets TBL1/β-catenin protein complex, suggesting BC-2059 as a therapeutic for tumors with deregulated Wnt signaling pathway. SIGNIFICANCE STATEMENT This study reports the mechanism of action of a novel Wnt pathway inhibitor, characterizing the selective disruption of the transducin β-like 1/β-catenin protein complex. As Wnt signaling is dysregulated across cancer types, this study suggests BC-2059 has the potential to benefit patients with tumors reliant on this pathway.

Background: Breast cancer (BCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in women. BCa is composed of distinct molecular subtypes, such as ER positive BCa (ER+ BCa) and triple negative BCa (TNBC). Development of novel effective therapies for patients with therapy resistant breast cancer (TR-BC) and TNBC remains the highest unmet need in patient treatment and survivorship. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) plays a critical role in multiple nuclear receptor functions leading to TR-BC and TNBC progression. PELP1 expression is dysregulated in BCa, is a prognostic indicator of poorer BCa survival, and its deregulation contributes to BCa therapy resistance. The objective of this study is development and characterization of a small molecule inhibitor of PELP1 (SMIP) as novel therapeutic for treating BCa. Methods: Using yeast two-hybrid screening, we identified PELP1 Inhibitory Peptide (PIP1) from a library of peptides. PIP1 binds PELP1 with high affinity and functions to inhibit PELP1 oncogenic activity. Direct binding of PIP1 to PELP1 was confirmed using biotin pull-down assays and inhibition of BCa proliferation confirmed using MTT assays. We used the Hit-Ligand interaction site with PIP1 hot spot residues based on 3D alignment and morphology to generate a library of peptidomimetics (small chemical molecules). In vitro activity was assessed using Celltiter Glo, MTT, and matrigel invasion chamber assays in multiple BCa models. Mechanistic studies were conducted using Western blot, reporter gene assays, and peptide competition assays. Xenograft and patient derived explant (PDEX) assays were used for preclinical evaluation and preliminary toxicity analysis. Results: Bioactivity screens revealed PELP1 Inhibitory Peptide (PIP1) significantly attenuates PELP1-mediated proliferation with an IC50 of 10µM across multiple BCa cell lines. We confirmed PIP1 binding to PELP1 using peptide pull-down assays with nuclear lysates from BCa cells. Using Hit-Ligand-Based interaction site with the PIP1 hot spot residues, we identified 61 potential hits using a 10,000 Diverse Set. Screening of these 61 potential hits using the MTT assays lead to the selection of SMIP34 (tetrahydropyrazolo [1,5a) pyrazole) as lead inhibitor of PELP1. SMIP34 treatment reduced proliferation at an IC50 of 3-10µM in ER+ BCa (ZR-75, MCF-7, and T- 47D); TR-BC (MCF-7-TamR, MCF-7-LTLT, ZR-75-MT-ER537S, and ZR-75-MT-ER538G); and TNBC (MDA- MB-231, and BT549) models. Additionally, SMIP34 showed no activity in human mammary epithelial cells. Specificity of SMIP34 was confirmed using PELP1 knockdown BCa cell lines. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. In matrigel invasion chamber assays, SMIP34 significantly reduced the invasiveness of TR-BC and TNBC models. In combination studies, SMIP34 displayed synergy and enhanced the efficacy of current chemotherapeutics Cisplatin and Paclitaxel. In PDEX assays, Ki67 staining revealed SMIP34 significantly decreased tumor proliferation. In xenograft models, SMIP34 (10mg/kg/s.c.) treatment resulted in significant reduction in tumors compared to vehicle treatment. Furthermore, overall mouse body weight in both control and SMIP34 treated groups were similar, suggesting no overt signs of toxicity. Conclusion: We have developed a first-in-class small molecule inhibitor of PELP1 (SMIP) displaying effectivity against TR-BC and TNBC in vitro and in vivo. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) Citation Format: Kristin A Altwegg, Suryavathi Viswanadhapalli, Monica Mann, Uday P Pratap, Mengxing Li, Junhao Liu, Yiliao Luo, Gangadhara R Sareddy, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Development and characterization of a first-in-class small molecule inhibitor of PELP1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-01.

BackgroundPatients with PD-L1-positive metastatic triple-negative breast cancers (mTNBC) who have been treated with atezolizumab+nab-paclitaxel had a clinically meaningful overall survival extension of 9.5 months compared to nab-paclitaxel alone, although overall survival in overall population was not statistically significant. Unlike many other cancers, immunotherapy for breast cancer has had limited success, due to the fact that there are very few T cells in the tumor microenvironment of mTNBC patients. Identifying ways to boost immunotherapy responses could change the paradigm of mTNBC, a disease still difficult to treat. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the tumors, leads to depletion of nutrients such as functional/natural amino acids. This metabolically stressful milieu causes activation of nutrient stress pathways, autophagy, and repressed immune responses. A key meditator of this nutrient stress pathway is a cytoplasmic Ser/Thr protein kinase called General Control Nonderepressible 2 (GCN2), also called EIF2AK4. GCN2 switches on following reduction of amino acids, and its activity results in T cell inactivation, T cell death, regulatory T cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs).MethodsWe have developed and synthesized a series of novel small molecule immunotherapeutic agents that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit pharmacological responses in immune cells and in breast cancer cells.ResultsGCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF-4 phosphorylation inhibition assays were performed. We confirmed on-target efficacy and tested the potency of our lead GCN2 inhibitor HCI-1046. HCI-1046 demonstrated potent activity, with an IC50 of 36 nM in inhibiting GCN2 kinase and exhibited cellular efficacy with an IC50 of 0.1 to 1.0 μM range. Our preliminary results support the hypothesis that the inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in breast cancer models. In vivo PK studies of HCI-1046 in rodents showed excellent PK properties; 55% oral bioavailability, low clearance, and >5 hour half-life.ConclusionsThus, HCI-1046 is nominated as a pre-clinical agent. Additional data regarding evaluation of the effects of HCI-1046 on the MDSC-suppressive function on T cells using ELISpot assays with breast cancer patient samples, and mouse model efficacy studies will be discussed.ReferencesEkiz HA, Lai SA, Gundlapalli H, Haroun F, Williams MA, Welm AL. Inhibition of RON kinase potentiates anti-CTLA-4 immunotherapy to shrink breast tumors and prevent metastatic outgrowth. Oncoimmunology 2018;7(9):e1480286.Toogood PL. Small molecule immuno-oncology therapeutic agents. Bioorg Med Chem Lett 2018;28(3):319–329.Ravindran R, Loebbermann J, Nakaya HI, Khan N, Ma H, Gama L, Machiah DK, Lawson B, Hakimpour P, Wang YC, Li S, Sharma P, Kaufman RJ, Martinez J, Pulendran B. The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation. Nature 2016;531(7595):523–527.Brazeau JF, Rosse G. Triazolo[4,5-d]pyrimidine derivatives as inhibitors of GCN2. ACS Med Chem Lett 2014;5(4):282–3.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked inherited enzymopathic disorder that may lead to transfusion-requiring acute hemolytic anemia (AHA) triggered by fava beans ingestion, infection or some drugs. The gene encoding for G6PD carries a large number of genetic variants that have varying pathogenicity. We reported on three G6PD variants in the Gaza Strip Palestinian population with differing clinical impacts and frequencies: G6PD Mediterranean

The central role of

Purpose: About 500,000 people each year worldwide die from metastatic breast cancer. More than 70% of these metastatic breast cancer patients have bone metastases and median survival time from diagnosis of bone metastasis is only 2-3 years. As breast cancer bone metastases are primarily osteolytic, patients suffer from significant morbidities, including severe pain, pathological fractures, nerve compression, and hypercalcemia. Previously, it was shown that RON receptor tyrosine kinase mediates breast cancer outgrowth at metastatic sites, promotes osteolytic bone destruction, and induces systemic immune suppression. We hypothesized that inhibition of RON in specialized resident macrophages and/or osteoclasts will eliminate metastasis through dual effects of blocking osteolysis and enhancing anti-tumor immunity. In this study, we have developed several bis(phosphonate) or phosphate conjugates of BMS-777607 and LY2801653, and report their accumulation in the bone microenvironment along with effects on osteolysis and tumor burden. Methods: We have utilized novel synthetic schemes for synthesis of the drug conjugates. In vitro kinase assays were performed using ADP-Glo RON kinase assay kit (Promega). Hydroxyapatite binding assay was used to determine binding of drug conjugates to the bone mineral. Changes in phosphorylation of RON was demonstrated by Western blotting. Plasma pharmacokinetics and bone accumulation of lead drug conjugates in mice was assessed by mass-spectrometry. An intratibial bone metastasis model in which MSP-expressing MMTV-PyMT tumor cells are injected into the tibia of mice was used to assess the effect of treatment with lead drug conjugates. Results: Seven novel drug conjugates were synthesized and inhibited RON kinase activity with an IC50 of 0.060-2.4 µM. Three drug conjugates demonstrated better hydroxyapatite binding when compared with BMS-777607. In agreement with the in vitro kinase activity, a variable decrease in the phosphorylation of RON was observed in cells treated with the drug conjugates. ZB-28, a monophosphonate derivative of BMS-777607, showed higher accumulation in the bone when compared with BMS-777607 but failed to block tumor-mediated bone osteolysis in an intratibial bone metastasis mouse model. Currently, a cleavable phosphate conjugate of BMS-777607 (ZB-32) is undergoing testing in the same mouse model. We have confirmed that ZB-32 and its hydrolyzed product ZB-33 accumulate in the bone to a significantly higher level than BMS-777607. These results along with preliminary ADME will be presented. Conclusion: Targeting RON kinase in the bone microenvironment is a promising approach to alleviate morbidities observed in breast cancer patients with bone metastases. We present novel bone-targeting conjugates of BMS-777607 and highlight the potential for clinical development. Citation Format: Trason Thode, Jaime Fornetti, Ryan Rodriguez del Villar, Alexis Weston, Serina Ng, Srikanta Dana, Raffaella Soldi, Mohan Kaadige, Hariprasad Vankayalapati, Srinivas Kasibhatla, Alana Welm, Sunil Sharma. Development of bone-targeted Ron inhibitors to treat osseous metastases from breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3948.

A significant proportion of estrogen receptor positive breast cancers (ER+BC) will initially respond to treatment, but many eventually develop therapy resistance (TR-BC), and progress to incurable metastases. Oncogenic ER coregulators overexpressed in BC can contribute to constitutive, ligand-independent and ligand-dependent signaling which drives growth, resistance to therapy and metastasis. Proline-, glutamic acid, and leucine-rich protein 1 (PELP1), is a known coregulator that plays a critical role in ER oncogenic functions. Its expression is deregulated in BC and is a prognostic indicator of poor BC survival. The lack of a small molecule inhibitor that directly targets PELP1 represents a major knowledge gap. Therefore, we conducted a large scale peptide library screening and identified novel Peptide Inhibitor of PELP1 (PIP1). We demonstrated that PIP1 directly interacts with PELP1, promotes its degradation and has the potential to block PELP1 oncogenic functions in vitro. Using innovative peptidomimetic technology, we modeled PIP1 and synthesized several derivatives as Small Molecule Inhibitors of PELP1 (SMIPs). Using MTT assay and multiple BC cell lines, we identified a lead compound, SMIP34 with an IC50 of 5-10µM and with minimal effect on human mammary epithelial cells. SMIP34 in vitro activity was assessed by colony formation and Matrigel invasion assays. Knockdown of PELP1 using shRNA in BC cells significantly reduced SMIP34 activity, indicating target specificity. Further, MST and biotin-SMIP34 pulldown confirmed direct binding of SMIP34 to PELP1. Using ER+WT, mtER (mutant ER), and TR-BC cell lines we demonstrated that SMIP34 exhibits antiproliferative effects and reduces invasiveness. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation via the proteasome pathway. Thus MG132 treatment attenuated SMIP34 mediated degradation. RTqPCR analyses confirmed SMIP34 treatment reduced expression of PELP1 target genes. RNAseq analyses showed SMIP34 treatment altered the expression of genes associated with Estrogen response, Cell cycle and Apoptosis pathways. Cell cycle analyses revealed SMIP34 treatment promoted S phase arrest of BC cell lines. Using ER+WT, mtER, and PDX tumor tissues ex vivo, we demonstrated that SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. Further, SMIP34 (20mg/kg/IP) treatment in vivo significantly reduced tumor progression in mouse models of ER+WT, mtER, and mtER patient-derived xenograft BC. Our results using in vitro, ex vivo, and in vivo models showcase SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling and may serve as a potential therapeutic molecule for treating ER+, mtER, and TR-BC. Supported by NIH 1F31CA257298 (KA) and VA I01BX004545 (RV). Citation Format: Kristin Ann Altwegg, Monica Mann, Dimple Chakravarty, Zexuan Liu, Junhao Liu, Uday P. Pratap, Behnam Ebrahimi, John R. Sanchez, Ben H. Park, Hariprasad Vankayalapati, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. A novel small molecule targeting oncogenic PELP1 demonstrates anti-tumor activity in wild-type and mutant ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 648.

Introduction/Purpose: The most common leukemia in adults is Acute myeloid leukemia (AML) and the 5-year overall survival (OS) in all AML patients is approximately 24%. The highest of AML deaths in the US alone is among older patients at ages ≥ 65 emphasizing the need for better therapeutics. The older patients have more limited curative options available due to their inability to tolerate aggressive chemotherapy. Identifying ways to boost immunotherapy responses could change the paradigm of AML, a disease still difficult to treat despite several approved targeted immunotherapeutic. The development of effective small molecules would be significant since small molecules not only target immunosuppressive mechanisms like mAbs but can stimulate intracellular pathways where antibodies are unable to permeate. In addition, small molecules can provide optimal pharmacokinetics and pharmacodynamics for oral administration, amenable clinical dosing, and can induce relatively acute antitumor efficacy to avoid systemic immunogenicity, thus providing a superior therapeutic potential. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the AML leads to depletion of nutrients such as functional/natural amino acids. A key meditator of this nutrient stress pathway, the cytoplasmic GCN2 (EIF2AK4) kinase, switches on reduction of amino acids, and this activity results in T-cell inactivation, T-cell death, regulatory T-cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs). We have discovered a series of novel small molecule immunotherapeutic agents (HCI-1046) that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit responses in AML patient samples and cell lines. Experimental Procedures: GCN2 cell-free kinase binding, EIF2α family selectivity, pGCN2 inhibition assays were performed and confirmed its on-target efficacy and potency of lead inhibitor HCI-1046. Additional experiments were conducted including CellTiterGlo, IncuCyte Live Cell Imaging, FACS Flow Cytometry, Cytotoxicity, Western Blot, ELISpot, and ELISA assays. Results and Summary: GCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF4 inhibition data confirmed on-target activities of our lead GCN2 inhibitor HCI-1046. HCI-1046 demonstrated potent activity, with an IC50 of 36 nM in inhibiting GCN2 kinase, and exhibited cellular efficacy with an IC50 of 0.1 to 0.5 μM range. Our preliminary results support the hypothesis that the inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in the AML cancer model in vivo. Our in vivo PK studies on HCI-1046 in rodent species showed excellent PK properties with 55% oral bioavailability, low clearance, and >5.0-hour half-life. Thus, HCI-1046 is nominated as a pre-clinical agent. Additional data regarding the evaluation of the effects of HCI-1046 on the MDSC-suppressive function on T-cells using ELISpot, ELISA assays in AML patient samples, and other mouse model efficacy studies results will be discussed. Citation Format: Zhaoliang Li, Kyle Medley, Dongqing Yan, Kimberly Coffman, Tony Pomicter, David Lum, David J. Bearss, Hariprasad Vankayalapati. Targeting GCN2 kinase-driven stress response inactivation to restore immunity in AML Cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB090.

Background RON (Recepteur d'Origine Nantais) receptor tyrosine kinase is a promising target for anti-cancer therapeutics. The aim of this study was to identify new RON inhibitors using virtual screening methods. Methods To this end, a ligand-based virtual screening approach was employed for screening of ZINC database on the homology model of RON receptor. All the selected hits were inspected in terms of drug-likeness, ADME properties, and toxicity profiles. Ligand-based similarity searches along with further filtering criteria led to the identification of two compounds, TKI1 and TKI2 that were evaluated using in vitro cell-based RON inhibition assays. Results The results showed that TKI1 and TKI2 could reduce phosphorylation of RON. Both compounds showed inhibitory activity of the downstream mTOR pathway with no apparent effects on other signaling mediators in a dose-dependent manner. Conclusion These compounds can provide a basis for developing novel anti-RON inhibitors applicable to cancer therapy using medicinal chemistry-oriented optimization strategies.

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

Chromatin remodeling SWItch/Sucrose-NonFermentable (SWI/SNF) complexes, initially identified in yeast 20 years ago, are evolutionarily conserved multi-subunit protein complexes that use the energy from hydrolysis of adenosine triphosphate (ATP) to remodel nucleosome structure and modulate transcription. Mutations in proteins of SWI/SNF complexes occur in 20% of human cancers including ovarian cancer (OC). Approximately 50% of ovarian clear cell carcinoma (OCCC) carries mutations in the SWI/SNF subunit ARID1A while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is driven primarily by genetic inactivation of the SWI/SNF ATPase SMARCA4 (BRG1) alongside epigenetic silencing of the homolog ATPase SMARCA2 (BRM). Dual loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A) which regulates the chromatin landscape and gene expression by demethylating proteins, including histone H3. LSD1 associates with epigenetic complexes such as the nucleosome remodeling deacetylase complex (NuRD) and SWI/SNF to inhibit the transcription of genes involved in tumor suppression and cell differentiation. TGCA analysis of human cancers shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. Further, SCCOHT and OCCC cell lines show low nM IC50s for the reversible LSD1 inhibitor SP-2577 (Seclidemstat, currently in clinical phase I trials), supporting that these SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Recently, it has been also shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of Endogenous Retroviruses Elements (ERVs) and may thereby overcome resistance to checkpoint blockade. Additionally, SCCOHTs have been shown to exhibit an immune-active tumor microenvironment with PD-L1 expression in both tumor and stromal cells that strongly correlated with T cell infiltration. Thus, in this study we investigated the ability of SP-2577 to promote anti-tumor immunity and T cell infiltration in SWI/SNF-mutant SCCOHT and OCCC models. Our data shows that the reversible LSD1 inhibitor SP-2577 stimulates IFN-dependent anti-tumor immunity in SCCOHT cells in vitro in a 3D immune-organoid platform. Additionally, SP-2577 promoted the expression of PD-L1 in both SCCOHT and OCCC models. Together our findings suggest that SP-2577 and checkpoint inhibitors as a therapeutic combination may induce or augment immunogenic responses in these tumors.

N-Glycanase deficiency, or NGLY1 deficiency, is an extremely rare human genetic disease. N-Glycanase, encoded by the gene NGLY1, is an important enzyme involved in protein deglycosylation of misfolded proteins. Deglycosylation of misfolded proteins precedes the endoplasmic reticulum (ER)-associated degradation (ERAD) process. NGLY1 patients produce little or no N-glycanase (Ngly1), and the symptoms include global developmental delay, frequent seizures, complex hyperkinetic movement disorder, difficulty in swallowing/aspiration, liver dysfunction, and a lack of tears. Unfortunately, there has not been any therapeutic option available for this rare disease so far. Recently, a proposed molecular mechanism for NGLY1 deficiency suggested that endo-β-N-acetylglucosaminidase (ENGase) inhibitors may be promising therapeutics for NGLY1 patients. Herein, we performed structure-based virtual screening utilizing FDA-approved drug database on this ENGase target to enable repurposing of existing drugs. Several Proton Pump Inhibitors (PPIs), a series of substituted 1H-benzo [d] imidazole, and 1H-imidazo [4,5-b] pyridines, among other scaffolds, have been identified as potent ENGase inhibitors. An electrophoretic mobility shift assay was employed to assess the inhibition of ENGase activity by these PPIs. Our efforts led to the discovery of Rabeprazole Sodium as the most promising hit with an IC50 of 4.47±0.44μM. This is the first report that describes the discovery of small molecule ENGase inhibitors, which can potentially be used for the treatment of human NGLY1 deficiency.

Background: Breast cancer (BC) is composed of distinct molecular subtypes, such as ER+ BC and triple negative BC (TNBC). Development of novel effective therapies for patients with TNBC remains the highest unmet need in patient treatment and survivorship. PELP1 plays an essential role in several pathways including hormonal signaling, cell cycle progression, ribosomal biogenesis, and DNA damage response. PELP1 expression is an independent prognostic predictor of shorter BC-specific survival and an independent prognostic factor for predicting poor survivorship in TNBC patients. Recently, we generated a small molecule inhibitor of PELP1 (SMIP34) that binds and inhibits PELP1 oncogenic signaling. The objective of this study is to test the utility of SMIP34 as a novel therapeutic for treating TNBC. Methods: We have selected seven TNBC model cell lines (BT549, MDA-MB-453, MDA-MB-231, MDA-MB-468, SUM-159, HCC1806, and HCC1937) and one human mammary epithelial (HMEC) cell line in this study. In vitro activity was assessed using Cell Titer Glo, MTT, colony formation, and matrigel invasion assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq. Xenograft tumor-derived explant (XDEX) assays and patient-derived tumor explant (PDEX) assays were used for preclinical evaluation. Results: Using a panel of BC model cell lines, we found that SMIP34 treatment reduced cell viability with an IC50 of 3-8µM with no activity in HMEC cells. Knockdown of PELP1 using shRNA in BC cells significantly reduced SMIP34 activity, confirming its target specificity. We confirmed the physical interaction of SMIP34 to PELP1 using biotin-SMIP34 and MST assays. SMIP34 treatment significantly reduced the invasiveness and colony formation of TNBC cell lines. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. Further, RTqPCR analyses confirmed SMIP34 treatment reduced expression of PELP1 target genes. Western analyses confirmed SMIP34 treatment significantly reduced known PELP1 downstream signaling. Mechanistic studies using global RNA-seq identified that SMIP34 treatment alters known PELP1 modulated pathways (ribosomal biogenesis). Using MDA-MB-231 xenograft and PDX tumor tissues in explant assays, we demonstrated that SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. Accordingly, in xenograft models, SMIP34 (20mg/kg/IP) treatment resulted in a significant reduction in tumor volume compared to vehicle control. Conclusion: Our results using in vitro, ex vivo, and in vivo studies demonstrated that the PELP1 inhibitor SMIP34 has therapeutic efficacy against TNBC. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545 (R.K.V) Citation Format: Kristin A. Altwegg, Suryavathi Viswanadhapalli, Junhao Liu, Zexuan Liu, Uday P. Pratap, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Evaluation of a novel PELP1 inhibitor for treatment of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1333.

In patients with metastatic castration-resistant prostate cancers (mCRPC) who have previously been treated with abiraterone or enzalutamide, the median overall survival is only 14 months. Unlike many other cancers, immunotherapies have had limited successes, due to the fact that there are very few T cells in the tumor microenvironment of prostate cancer (PC) patients. Identifying ways to boost immunotherapy responses could change the paradigm of mCRPC, a disease still difficult to treat. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the tumors, leads to depletion of nutrients such as functional/natural amino acids. This metabolically stressful milieu causes activation of nutrient stress pathways, autophagy, and repressed immune responses. A key meditator of this nutrient stress pathway is a cytoplasmic Ser/Thr protein kinase called General Control Nonderepressible 2 (GCN2), also called EIF2AK4. GCN2 switches on by a reduction of amino acids, and its activity results in T cell inactivation, T cell death, regulatory T cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs). We have developed and synthesized a series of novel small molecule immunotherapeutic agents that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit pharmacological responses in immune cells. GCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF-4 phosphorylation inhibition assays were performed and confirmed it's on-target efficacy and potency of lead GCN2 inhibitor HCI-1046. In these studies, our lead GCN2 kinase inhibitor HCI-1046 demonstrated potent activity with an IC50 of 36 nM in inhibiting GCN2. GCN2 expression has been detected in PC-3, DU-145, and LNCap cell lines, and HCI-1046 exhibited cellular efficacy with an IC50 of 0.9 to 8 μM range, reduced phosphorylation of GCN2, ATF-4 significantly and its downstream target eIF2α. HCI-1046 inhibited pGCN2 and pEIF2α in human prostate cancer clinical patient samples derived MDSC cultured in amino-acid starved conditions. HCI-1046, in a dose-dependent fashion, restored CD8+ T cell proliferation and function in clinical samples of PC patients. Our preliminary results support the hypothesis that inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in PC cellular models. In vivo PK studies of HCI-1046 in rodent species showed excellent PK properties; 55% oral bioavailability, low clearance, and >5 hours half-life. HCI-1046 is nominated as a pre-clinical agent. Additional 3D cellular efficacy studies, FACS for cellular apoptosis, cell migration, live PC cells including immuno-ELISA, ELISpot experimental results for MDSCs suppressive function of T cells, and restoration by HCI-1046 will be presented at the conference. PC mouse model efficacy studies will also be discussed. Citation Format: Kyle Medley, Zhaoliang Li, Dongqing Yan, Umang Swami, Neeraj Agarwal, Hariprasad Vankayalapati. Targeting GCN2 kinase-driven stress response inactivation by orally available small molecules to restore immune tumor microenvironment in prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB136.

Background: Mutations in ESR1 genes (30-40% frequency) play an important role in acquired endocrine therapy resistance and metastases. The most commonly observed are two ESR1 LBD mutations, D538G andY537S. These mutant ERα (MT) proteins have high constitutive transcriptional activity leading to therapy resistance. Furthermore, the ability of the constitutively active mutants to interact with coregulators is associated with the promotion of tumor growth. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1), an oncogenic coregulator of ERα, plays a critical role in ERα signaling, and its dysregulated expression is a prognostic indicator for poorer breast cancer (BCa) survival. The objective of this study was to test the utility of Small Molecule Inhibitor of PELP1 (SMIP34) for treating ESR1 mutant (MT-ER) driven BCa. Methods: Four BCa models that express either ESR1 mutation D538G or Y537S and their respective wild-typeERα (WT-ER) controls were used to test the utility of targeting the PELP1 axis using PELP1-specific shRNA orSMIP34. Celltiter Glo, MTT, colony formation, and Boyden chamber invasion assays were used to test the efficacy of SMIP34. Western blot, RNA-Seq, and reporter gene assays were utilized to uncover the mechanistic insights. Pre-clinical evaluation was performed using MT-ER expressing xenograft explant (XDEX) and patient-derived explant (PDEX) assays. Results: BCa model cells expressing MT-ER showed increased cell proliferation, whilst PELP1 knock-down significantly reduced their proliferation. Immunoprecipitation results confirmed that PELP1 constitutively associates with MT-ER. PELP1 knock-down or treatment with PELP1 inhibitor SMIP34 significantly reduced proliferation of the four MT-ER models with an IC50 of 3-5μM. PELP1 knock-down or SMIP34 treatment significantly reduced the constitutive ERE reporter activity observed in MT-ER models. RTqPCR assays confirmed the downregulation of MT-ER target genes in PELP1 knock-down and SMIP34 treated cells. Furthermore, PELP1 knock-down or SMIP34 treatment significantly reduced the invasiveness and colony formation of MT-ER BCa models. Mechanistic studies using Western blot revealed that SMIP34 contributes to PELP1 degradation by its direct binding to PELP1. SMIP34 significantly decreased proliferation of MT-ER BCa cells in XDEX andPDEX assays, as measured by Ki67 staining. Conclusion: Our results suggest that PELP1 associates with MT-ER and targeting the PELP1 axis with SMIP34will have therapeutic utility in treating MT-ER driven BCa. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545(R.K.V) Citation Format: Kristin A Altwegg, Suryavathi Viswanadhapalli, Junhao Liu, Zexuan Liu, Uday P. Pratap, Benham Ebrahimi, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Targeting the PELP1 axis for treating ESR1 mutant driven breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-15.

BACKGROUND: Evolving evidence suggests that cancer stem cells (CSCs) evade hormonal therapy and therapy resistance occurs due to regrowth of tumor cells from cancer stem cells that escaped hormonal therapy or remained in the body after tumor resection. Recent studies suggest that estrogen stimulates breast cancer stem-cells and G9a/EHMT2 plays a critical role in stem cell maintenance. Proline, glutamic acid, and leucine rich protein (PELP1) is a proto-oncogene that functions as a critical coregulator of several nuclear receptors and other transcription factors. PELP1 is commonly overexpressed in hormone-related cancers, and is prognostically linked to shorter breast cancer survival. Recent studies from our lab discovered PELP1 interacts with G9a/EHMT2. However, it remains unknown whether PELP1-G9a signaling plays a role in breast cancer stem cell proliferation. The objective of this study is to develop small molecular inhibitors that block G9a/EHMT2 interactions and to test their utility. METHODS: We isolated CD44high/CD24low CSCs from three breast cancer cell lines (ZR75, MCF7, T47D) using FACS. To test the effect of PELP1 inhibitors on CSCs, we cultured CSCs in SFM in the presence or absence of PELP1 inhibitors for a period of 7-10 days. Cells were analyzed for spheroid formation, morphological changes, immunofluorescence for differentiation markers, protein (Western) and RNA (RT-qPCR) analysis. Expression of differentiation markers K19 and K14 and stem cell markers CD133, CD44, Id1, Nestin, Musashi-1, SOX2, Notch2, and OCT1 was determined. RESULTS: Using mapping studies, we identified a small peptide inhibitor (PIP1) that interferes PELP1 interaction with G9/EHMT2. Utilizing Hit-Ligand interaction site with the PELP1 hot spot residues based on 3D alignment and shape, we have identified 61 potential hits from Ligand-Based screening using a 10,000 Diverse Set. Screening of these 61 potential hits using MTT based cell viability assays identified three small organic molecule inhibitors (peptidomimetics) as leads. All three peptidomimetics (#20, #29, #34) showed activity similar to PELP1 peptide inhibitor 1 (PIP1) in assays using three different breast cancer cell lines. Further, PELP1 targeting peptidomimetic disrupted PELP1 interaction with G9a/EHMT2. Peptidomimetic treatment inhibited the proliferation of tamoxifen therapy resistant cells. In mechanistic studies, we found that knockdown of PELP1 inhibited stem cell maintenance. In FACS analysis of ZR75, ZR75-PELP1 and ZR75-PELP1KD cells, the percentage of CD44high/CD24low cells correlated with PELP1 status. Accordingly, in mammosphere formation assays, PELP1 targeting peptidomimetic significantly inhibited the formation of mammospheres and the size of the mammospheres was also substantially decreased. Further, in self-renewal assays, peptidomimetic-treated cells had decreased self-renewal capacity. CONCLUSIONS: Collectively, our studies have discovered an essential role for PELP1 in breast cancer stem cell maintenance and identified the PELP1- G9a/EHMT2 axis as a potential therapeutic target for reducing stemness. Further, the novel small molecule inhibitors of PELP1 could be used for therapeutic targeting of breast cancer stem cells and therapy resistance. Citation Format: Viswanadhapalli S, Mann M, Sareddy GR, Xaionan L, Vankayalapati H, Brann D, Vadlamudi RK. Proto-oncogene PELP1 signaling regulates breast cancer stem cells via G9a/EHMT2. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-06-09.

Background: Breast cancer (BC) is the most commonly diagnosed cancer and the second leading cause of cancer death in American women. BC is composed of distinct molecular subtypes, such as ER positive BC (ER+) and triple negative BC (TNBC). Development of novel effective therapies for patients with therapy resistant BC (TR-BC) and TNBC remains the highest unmet need in patient treatment and survivorship. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1) plays a critical role in multiple nuclear receptor functions leading to TR-BC and TNBC progression. PELP1 expression is dysregulated in BC, is a prognostic indicator of poorer BC survival, and its deregulation contributes to TR-BC. The objective of this study is characterization of a small molecule inhibitor of PELP1 (SMIP34) as novel therapeutic for treating advanced BC. Methods: Yeast two-hybrid screening was used to identify PELP1 Inhibitory Peptide 1 (PIP1). Direct binding of PIP1 to PELP1 was confirmed using biotin pull-down assays and inhibition of BC proliferation confirmed using MTT assays. Hit-Ligand interaction site with PIP1 hot spot residues based on 3D alignment and morphology was used to generate a library of small chemical molecules that function as peptidomimetics of PIP1. In vitro activity was assessed using CellTiter Glo, MTT, colony formation and matrigel invasion assays in multiple BC models. Mechanistic studies were conducted using Western blot, and reporter gene assays. Xenograft and patient derived explant (PDEX) assays were used for preclinical evaluation. Results: Screening utilizing MTT assays lead to the selection of SMIP34 as an inhibitor of PELP1. SMIP34 treatment reduced cell viability at an IC50 of 3-10µM in a panel of BC cells. Additionally, SMIP34 showed no activity in human mammary epithelial cells. Knockdown of PELP1 in BC cells significantly reduced the SMIP34 activity, confirming target specificity. Furthermore, SMIP34 treatment significantly reduced the invasiveness and colony formation of TR-BC and TNBC cells. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. In combination studies, SMIP34 displayed synergy and enhanced the efficacy of current chemotherapeutics Cisplatin and Paclitaxel. In PDEX assays, SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. In xenograft models, SMIP34 (10mg/kg/s.c.) treatment resulted in significant reduction in tumor volume compared to vehicle control. Furthermore, overall mouse body weight in both control and SMIP34 treated groups were similar, suggesting no overt signs of toxicity. Conclusion: We have developed a first-in-class small molecule inhibitor of PELP1 (SMIP34) displaying therapeutic efficacy against TR-BC and TNBC in vitro, ex vivo, and in vivo. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545 (R.K.V) Citation Format: Kristin Ann Altwegg, Suryavathi Viswanadhapalli, Monica Mann, Uday P. Pratap, Mengxing Li, Junhao Liu, Yiliao Luo, Gangadhara R. Sareddy, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Characterization of small molecule inhibitors of PELP1 for treating advanced breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6403.

Genomic instability is a recognized hallmark of cancer. Germline mutations in critical DNA-repair and DNA-damage response genes predispose to cancer development, but also create vulnerabilities that can be exploited for cancer therapy. Poly (ADP-ribose) polymerase (PARP) inhibitors are selectively active in cells with homologous recombination deficiency (HRD) caused by mutations in BRCA1, BRCA2, and other DNA repair genes. PARP inhibitors elicit significant responses in ovarian and breast cancers from BRCA1 or BRCA2 mutation carriers. However, many cancers that initially respond to PARP inhibitors eventually develop drug resistance. Thus, it is important to develop new strategies to enhance PARP inhibitor sensitivity and to increase the duration of response. We have identified Salt Induced Kinase 2 (SIK2) inhibitors (ARN3236 and ARN3261) that induce double strand breaks (DSBs) in DNA of HR-competent cells and produce synthetic lethality with multiple PARP inhibitors. SIK2 is an AMP-activated protein kinase (AMPK)-related protein kinase that is required for ovarian cancer cell proliferation and metastasis. SIK2 is overexpressed and correlates with poor prognosis in patients with high-grade serous ovarian carcinoma and triple negative breast cancer. SIK2 inhibition enhances paclitaxel sensitivity in both cancer types. We have demonstrated that olaparib-induced-growth inhibition was significantly enhanced by concurrent treatment with either ARN3236 or ARN3261 in each of 12 ovarian and breast cancer cell lines tested, but not in 3 non-tumorigenic cell lines. Co-administration of olaparib with SIK2 inhibitors suppressed tumor growth and increased the survival of mice with human ovarian (OC316) and breast (MDA-MB-231) cancer xenografts without affecting animal weight. ARN3261 produced little toxicity in preclinical toxicology studies. SIK2 inhibitors decrease the phosphorylation of class-IIa HDAC4/5/7 and abolish class-IIa HDAC 4/5/7-associated transcriptional activity of Myocyte Enhancer Factor 2D (MEF2D). Genome-wide chromatin immunoprecipitation (CHIP) sequencing revealed that SIK2 inhibitors reduce MEF2D binding to regulatory regions with high-chromatin accessibility in DNA repair genes, including FANCD2, EXO1 and XRCC4, resulting in repression of critical genes in DNA DSB repair pathway and induction of apoptosis. In addition, SIK2 inhibitors significantly decreased olaparib-mediated PARP activity and enhanced olaparib-induced cytotoxicity. Together, our data argue that the combination of a SIK2 inhibitor and a PARP inhibitor has the potential to increase the magnitude and duration of PARP inhibitor activity with tolerable toxicity. Use of a SIK2 inhibitor in combination with a PARP inhibitor provides a novel therapeutic strategy for ovarian and triple negative breast cancers with or without BRCA gene mutation. Citation Format: Zhen Lu, Weiqun Mso, Janice M. Santiago-O'Farrill, Hailing Yang, Lan Pang, Ahmed A. Ahmed, Hariprasad Vankayalapati, Robert C. Bast Jr. Novel SIK2 inhibitors sensitize ovarian and breast cancer to PARP inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5210.

Xylosides are small molecules that serve as primers of glycosaminoglycan biosynthesis. Xyloside mediated modulation of biological functions depends on the extent of priming activity and fine structures of primed GAG chains. In earlier studies, copper (Cu) catalyzed synthesis of click-xylosides and their priming activity were extensively documented. In the current study, ruthenium (Ru) mediated catalysis was employed to synthesize xylosides with a 1,5-linkage between the xylose and the triazole ring instead of a 1,4-linkage as found in Cu-catalyzed click-xyloside synthesis. Mono- and bis-click-xylosides were synthesized using each catalytic method and their glycosaminoglycan priming activity was assessed in vitro using a cellular system. Ru-catalyzed click-xylosides showed a higher priming activity as measured by incorporation of radioactive sulfate into primed glycosaminoglycan chains. This study demonstrates that altering the linkage of the aglycone to the triazole ring changes the priming activity. Computational modeling provides a molecular rationale for higher priming ability of Ru-mediated click-xylosides. Higher GAG priming activity is attributed to the formation of more stable interactions between the 1,5-linked xylosides and β-1,4-galactosyltransferase 7 (β4GalT7).

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we develop two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable for high throughput screening. Using ETS1 as a model, these assays were applied to screen 110 compounds derived from a high-throughput virtual screen. We find that the use of lower affinity DNA-binding sequences, similar to those which ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

In this work, we describe the use of the rule of 3 fragment-based strategies from biochemical screening data of 1100 in-house, small, low molecular weight fragments. The sequential combination of in silico fragment hopping and fragment linking based on S160/Y161/A162 hinge residues hydrogen bonding interactions leads to the identification of novel 1 H -benzo[ d ]imidazol-2-yl)-1 H -indazol class of Phosphoinositide-Dependent Kinase-1 (PDK1) inhibitors. Consequent SAR and follow-up screening data led to the discovery of two potent PDK1 inhibitors: compound 32 and 35 , with an IC 50 of 80 nM and 94 nM, respectively. Further biological evaluation showed that, at the low nanomolar concentration, the drug had potent ability to inhibit phosphorylation of AKT and p70S6, and selectively kill the cancer cells with mutations in both PTEN and PI3K. The microarray data showed that DUSP6, DUSP4, and FOSL1 were down-regulated in the sensitive cell lines with the compound treatment. The in vivo test showed that 35 can significantly inhibit tumor growth without influencing body weight growth. Our results suggest that these compounds, especially 35 , merit further pre-clinical evaluation.

Purpose: Small cell carcinoma of the ovary-hypercalcemic type (SCCOHT) is a rare ovarian cancer of young women characterized by SWI/SNF ATPase SMARCA4 (BRG1) genetic inactivation alongside epigenetic silencing of the homolog ATPase SMARCA2 (BRM). Because the SWI/SNF complex associates with histone modifying complexes (HMCs) containing targetable epigenetic enzymes, the lack of SWI/SNF ATPase activity is hypothesized to interrupt normal function of HMCs that result in altered epigenetic landscape. Lysine-specific histone demethylase 1 (LSD1 or KDM1) regulates the chromatin landscape and gene expression by removing specific methyl groups from methylated proteins including histone H3. It associates with a number of transcriptional regulatory complexes including SWI/SNF and acts in a context-dependent manner. We and others have found that LSD1 is among the most highly expressed histone modifiers in SCCOHT. SCCOHT cell lines are very sensitive to the reversible LSD1 inhibitor SP-2509 compared to other epigenetic agents like EZH2 and bromodomain inhibitors, suggesting that LSD1 dependence is a defining feature of these SWI/SNF-deficient ovarian cancers. Recently it has been shown that LSD1 inhibition stimulates an INF-dependent anti-tumor immunity and overcomes resistance to checkpoint blockade through induction of Endogenous Retroviruses (ERVs) in breast cancer. Interestingly, SCCOHT have been shown to exhibit an immune-active tumor microenvironment with PD-L1 expression in both tumor and stromal cells that strongly correlated with T-cell infiltration (TIL). In this study we investigate the ability of SP-2509 to promote anti-tumor immunity and T-cell infiltration in SCCOHT. Experimental Design: SCCOHT cell line BIN67, COV434 and SCCOHT1 were subjected to RT-PCR to evaluate ERVs induction and INF expression in response to SP-2509 treatment. SCCOHT cells were also co-cultured with peripheral blood mononuclear cells (PBMCs) in a 3D platform to evaluate T-cell infiltration following SP-2509 treatment. Results: SP-2509 promotes ERV-mediated immune response in SCCOHT cell lines. Additionally, SP-2509 stimulates T-cell infiltration in SCCOHT in a spheroid platform. Conclusion: Our data suggest that the reversible LSD1 inhibitor SP-2509 stimulates an INF-dependent anti-tumor immunity in SCCOHT cells in vitro and in 3D platform. Given that LSD-1 is a potential therapeutic target in SWI/SNF mutated SCCOHT, the combination of SP-2509 and PD-1 inhibitor represents a plausible combination to induce or augment immunogenic responses in these tumors. Citation Format: Raffaella Soldi, Alexis Weston, Trason Thode, Rhonda Lewis, Mohan Kaadige, Hariprasad Vankayalapati, William Hendricks, Sunil Sharma. The reversible LSD1 inhibitor SP-2509 promotes anti-tumor immunity in small cell carcinoma of the ovary-hypercalcemic type (SCCOHT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3869.

Lysine specific demethylase 1 (LSD1) plays an important role in regulating histone lysine methylation at residues K4 and K9 on histone H3 and is an attractive therapeutic target in multiple malignancies. Here we report a structure-based virtual screen of a compound library containing ∼2 million small molecular entities. Computational docking and scoring followed by biochemical screening led to the identification of a novel N'-(1-phenylethylidene)-benzohydrazide series of LSD1 inhibitors with hits showing biochemical IC50s in the 200-400 nM range. Hit-to-lead optimization and structure-activity relationship studies aided in the discovery of compound 12, with a Ki of 31 nM. Compound 12 is reversible and specific for LSD1 as compared to the monoamine oxidases shows minimal inhibition of CYPs and hERG and inhibits proliferation and survival in several cancer cell lines, including breast and colorectal cancer. Compound 12 may be used to probe LSD1's biological role in these cancers.

Purpose: Salt-inducible kinase 2 (SIK2) is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here, we examine the effects of a novel small-molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer. Experimental Design: SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear–centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death, and reduce AKT/survivin signaling. Results: SIK2 is overexpressed in approximately 30% of high-grade serous ovarian cancers. ARN-3236 inhibited the growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 μmol/L, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson r = −0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip (P = 0.028) and OVCAR8 xenografts. In at least three cell lines, a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest, and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression. Conclusions: ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity. Clin Cancer Res; 23(8); 1945–54. ©2016 AACR.

Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting more than 500 million people worldwide, is one of the most common of inherited disorders. There are 186 G6PD mutations published, with mutational clustering within defined ethnic/racial groups. However comprehensive molecular characterization of ethnically associated G6PD mutants and their clinical implications are lacking. Design and methods Eighty unrelated Palestinian children hospitalized for hemolysis were studied. G6PD activity was determined by quantitative spectrophotometry and G6PD mutations were analyzed by sequencing of gDNA. Results 65 of 80 children (81%) had G6PD deficiency, accounting for most of the hemolytic disease in this age group. G6PD Mediterraneanc.563T, African G6PD A-c.202A/c.376G, and G6PD Cairoc.404C were common with relative allele frequencies of 0.33 [1], 0.26, and 0.18 respectively. Two other variants were discovered, G6PD Beverly Hillsc.1160A mutation, and a novel G6PD missense mutation c.536G > A (Ser179Asn), designated G6PD “Gaza”. Three samples exhibited enzyme deficiency without detectable exonic or exon/intron boundary mutations. Conclusion G6PD deficiency accounts for the majority of diagnoses for hemolysis in Palestinian children (81%), providing support for newborn G6PD deficiency screening programs. We report unanticipated molecular heterogeneity of G6PD variants among Gaza Strip Palestinians greater than reported in neighboring Arab populations. We report a high proportion of affected children with G6PD Cairo, which was observed previously in only a single Egyptian, and a novel mutation G6PD “Gaza”.

Classic Galactosemia is an autosomal recessive disorder caused by the deficiency of galactose-1-phosphate uridylyltransferase (GALT), one of the key enzymes in the Leloir pathway of galactose metabolism. While the neonatal morbidity and mortality of the disease are now mostly prevented by newborn screening and galactose restriction, long-term outcome for older children and adults with this disorder remains unsatisfactory. The pathophysiology of Classic Galactosemia is complex, but there is convincing evidence that galactose-1-phosphate (gal-1P) accumulation is a major, if not the sole pathogenic factor. Galactokinase (GALK) inhibition will eliminate the accumulation of gal-1P from both dietary sources and endogenous production, and efforts toward identification of therapeutic small molecule GALK inhibitors are reviewed in detail. Experimental and computational high-throughput screenings of compound libraries to identify GALK inhibitors have been conducted, and subsequent studies aimed to characterize, prioritize, as well as to optimize the identified positives have been implemented to improve the potency of promising compounds. Although none of the identified GALK inhibitors inhibits glucokinase and hexokinase, some of them cross-inhibit other related enzymes in the GHMP small molecule kinase superfamily. While this finding may render the on-going hit-to-lead process more challenging, there is growing evidence that such cross-inhibition could also lead to advances in antimicrobial and anti-cancer therapies.

Receptor tyrosine kinases (RTK) are cell-surface transmembrane receptors that contain regulated kinase activity within their cytoplasmic domain and play an important role in signal transduction in both normal and malignant cells. The mammalian TAM RTK family includes 3 closely related members: Tyro-3, Axl, and Mer. Overexpression or ectopic expression of the TAM receptors has been detected in a wide array of human cancers. Growth arrest-specific gene 6 has been identified as the major ligand for these TAM RTKs, and its binding to the receptors has been shown to promote proliferation and survival of cancer cells in vitro. Abnormal expression and activation of Axl or Mer can provide a survival advantage for certain cancer cells. Inhibition of Axl and Mer may enhance the sensitivity of cancer cells to cytotoxic agents and would potentially be a therapeutic strategy to target cancer cells. This review elucidates the role of Axl and Mer in normal cellular function and their role in oncogenesis. In addition, we review the potential to inhibit these RTKs for the development of therapeutic targets in treatment of cancer. Mol Cancer Ther; 10(10); 1763–73. ©2011 AACR.

Objective Constitutive activation of the Janus kinase 2 (JAK2) due to a somatic mutation ( JAK2 V617F ) arising in hematopoietic stem cells plays a central role in the pathophysiology of myeloproliferative neoplasms (MPNs). To investigate the hypothesis that drugs that inhibit JAK2 have therapeutic potential, we developed a small molecule inhibitor, SGI-1252, that targets the adenosine triphosphate-binding and solvent pocket of the protein. Materials and Methods Established cells lines each expressing different JAK2 V617F copy numbers, a cell line transfected with wild-type and mutant JAK2 , ex vivo expanded erythroid progenitor cells from patients with MPNs, and a murine xenograft model were used to characterize the activity of SGI-1252. Results In vitro studies showed that SGI-1252 potently inhibits the kinase activity of wild-type JAK2, JAK2 V617F and JAK1, but not JAK3. SGI-1252 blocked phosphorylation of signal transducers and activators of transcription 5, a downstream target of JAK2 and inhibited expression of the JAK2-dependent antiapoptotic gene BCL-X L . Additional studies confirmed induction of apoptosis in JAK2 V617F -positive cell lines by SGI-1252. Moreover, cell lines transfected with either wild-type JAK2 or JAK2 V617F were equally susceptible to the antiproliferative effects of SGI-1252 and the antiproliferative activity of SGI-1252 toward ex vivo−expanded erythroid progenitors from patients with polycythemia vera and primary myelofibrosis appeared independent of the JAK2 V617F allele burden. Pharmacodynamic studies in a murine xenograft model demonstrated both anti-tumor activity and inhibition of signal transducers and activators of transcription 5 phosphorylation by SGI-1252, and the drug was active and well-tolerated whether delivered intraperitoneally or orally. Conclusions Together, these studies support further development of SGI-1252 for clinical use.

Genomic instability is a recognized hallmark of cancer. Germline mutations in critical DNA-repair and DNA-damage response genes, including BRCA1and BRCA2, predispose to cancer development, but also create vulnerabilities that can be exploited for cancer therapy. The successful development of PARP inhibitors for cancers with BRCA1/2 mutations and a deficiency in homologous recombination DNA repair has provided proof of concept. Inhibition of kinases that promote transcription of DNA repair genes might provide targets that enhance the activity of PARP-inhibitors. One novel candidate, salt-inducible kinase 2 (SIK2), is overexpressed in approximately 30% of high grade serous ovarian cancers. Inhibition of SIK2 induces tetraploidy, triggers apoptotic cell death, prevents metastasis, reduces AKT/survivin signaling and decreases phosphorylation of class-IIa histone deacetylases (HDACs) enhancing inhibition of gene expression. In contrast to other HDACs, class-IIa HDACs function as signal-responsive transcriptional corepressors for the myocyte enhancer factor-2 (MEF2) family of transcription factors. MEF2s play important roles in regulating growth factor responses, neuronal survival, T-cell apoptosis and, importantly, B-cell DNA double strand break (DSB) repair. Here we ask whether inhibition of SIK2 can enhance HDAC class-IIa inhibition of MEF activity, decrease DNA repair and enhance sensitivity to the PARP inhibitor Olaparib in ovarian cancer cells. Treatment with SIK2 inhibitors (ARN3236 or ARN3261) enhanced sensitivity to Olaparib in each of 8 ovarian cancer cell lines without BRCA1/2 mutations (OC316, OVCAR8, IGROV1, A2780, HCC5030, HCC5032, SKOv3 and OVCAR5) and significantly reduced the IC50 of Olaparib in two PARP inhibitor-resistant ovarian cancer cell lines (A2780CP-R and UWB1-289-10R). Synergistic cytotoxicity, judged with a Chou-Talaylay combination index (CI), was observed in all 8 cell lines. Treatment with a SIK2 inhibitor decreased the phosphorylation of class-IIa HDAC4/5/7, abolished class-IIa HDAC4/5/7-associated transcriptional activity of MEF2 and decreased MEF2 binding to regulatory regions with high-chromatin accessibility in DNA repair genes, resulting in repression of critical gene expression in DNA repair pathways. DNA damage, judged by rH2AX expression was enhanced by SIK2 inhibition. These observations not only provide new insights into the transcriptional regulation of DNA repair gene expression, but also have important implications for enhancing sensitivity of high grade serous ovarian cancers to PARP inhibition, with or without BRCA1 or BRCA2 germline mutations. Citation Format: Zhen Lu, Wequn Mao, Lan Pang, Janice M. Santiago-O'Farrill, Haling Yang, Ahmed Ahmed, Hariprasad Vankayalapati, Robert C. Bast. SIK2 inhibitors regulate DNA repair pathway and sensitize ovarian cancer to PARP1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 324.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease with multiple, distinct molecular subtypes that exhibit unique transcriptional programs and clinical progression trajectories. Despite knowledge of the molecular heterogeneity of the disease, most patients are limited to generic, indiscriminate treatment options: cytotoxic chemotherapy, surgery, and radiation. To identify new intervention targets in TNBC, we used large-scale, loss-of-function screening to identify molecular vulnerabilities among different oncogenomic backgrounds. This strategy returned salt inducible kinase 2 (SIK2) as essential for TNBC survival. Genetic or pharmacological inhibition of SIK2 leads to increased autophagic flux in both normal-immortalized and tumor-derived cell lines. However, this activity causes cell death selectively in breast cancer cells and is biased toward the claudin-low subtype. Depletion of ATG5, which is essential for autophagic vesicle formation, rescued the loss of viability following SIK2 inhibition. Importantly, we find that SIK2 is essential for TNBC tumor growth in vivo . Taken together, these findings indicate that claudin-low tumor cells rely on SIK2 to restrain maladaptive autophagic activation. Inhibition of SIK2 therefore presents itself as an intervention opportunity to reactivate this tumor suppressor mechanism.

The Wnt/β-catenin signaling pathway plays a vital role in cell growth, the regulation, cell development, and the differentiation of normal stem cells. Constitutive activation of the Wnt/β-catenin signaling pathway is found in many human cancers, and thus, it is an attractive target for anticancer therapy. Specific inhibitors of this pathway have been keenly researched and developed. Cell based screening of compounds library, hit-to-lead optimization, computational and structure-based design strategies resulted in the design and synthesis of a series of anthracene-9,10-dione dioxime series of compounds demonstrated potent inhibition of β-catenin in vitro (IC50 < 10 nM, 14) and the growth of several cancer cell lines. This article discusses the potential of inhibiting the Wnt/β-catenin signaling pathway as a therapeutic approach for cancer along with an overview of the development of specific inhibitors.

Aurora A and Aurora B are potential targets for anticancer drug development due to their roles in tumorigenesis and disease progression. To identify small-molecule inhibitors of the Aurora kinases, we undertook a structure-based design approach that used three-dimensional structural models of the Aurora A kinase and molecular docking simulations of chemical entities. Based on these computational methods, a new generation of inhibitors derived from quinazoline and pyrimidine-based tricyclic scaffolds were synthesized and evaluated for Aurora A kinase inhibitory activity, which led to the identification of 4-(6,7-dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioic acid [4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide. The lead compound showed selectivity for the Aurora kinases when it was evaluated against a panel of diverse kinases. Additionally, the compound was evaluated in cell-based assays, showing a dose-dependent decrease in phospho-histone H3 levels and an arrest of the cell cycle in the G2-M fraction. Although biological effects were observed only at relatively high concentrations, this chemical series provides an excellent starting point for drug optimization and further development. [Mol Cancer Ther 2006;5(7):1764–72]

Psorospermin is a natural product that has been shown to have activity against drug-resistant leukemia lines and AIDS-related lymphoma. It has also been shown to alkylate DNA through an epoxide-mediated electrophilic attack, and this alkylation is greatly enhanced at specific sites by topoisomerase II. In this article, we describe the synthesis of the two diastereomers of O5-methyl psorospermin and their in vitro activity against a range of solid and hematopoietic tumors. The diastereomeric pair (±)-(2′R,3′R) having the naturally occurring enantiomer (2′R,3′R) is the most active across all the cell lines and shows approximately equal activity in both drug-sensitive and drug-resistant cell lines. In subsequent studies using all four enantiomers of O5-methyl psorospermin, the order of biological potency is (2′R,3′R) > (2′R,3′S) = (2′S,3′R) > (2′S,3′S). This order of potency is also found in the topoisomerase II–induced alkylation of O5-methyl psorospermin and can be rationalized by molecular modeling of the psorospermin-duplex binding complex. Therefore, this study defines the optimum stereochemical requirements for both the topoisomerase II–induced alkylation of DNA and the biological activity by psorospermin and its O5-methyl derivatives. Finally, (2′R,3′R) psorospermin was found to be as effective as gemcitabine in slowing tumor growth in vivo in a MiaPaCa pancreatic cancer model. In addition, (2′R,3′R) psorospermin in combination with gemcitabine was found to show an at least additive effect in slowing tumor growth of MiaPaCa.

The antileukemic xanthone psorospermin is a topoisomerase II-dependent DNA alkylator in advanced preclinical development. Efforts have been made to further understand the structural requirements of its mechanism of action through the synthesis of ring-constrained analogues, based on the skeleton of the bisfuranoxanthone natural products. Molecules were designed that contain the bisfuran and xanthone portions of naturally occurring psorofebrins, and molecular modeling was used to assess their DNA alkylating potential and to refine the structures. A short, diastereoselective synthetic process to access bisfuranoxanthones was developed, culminating in the first total synthesis of (+/-)-isohydroxypsorofebrin. Two compounds designed and synthesized were of particular interest, chlorohydrin 7 and epoxide 6, which are reactive analogues of the natural product isohydroxypsorofebrin. The chlorohydrin retains the psorospermin-like DNA alkylation characteristics despite its rigid structure and high innate affinity for DNA. Molecular modeling has been used to rationalize the increased activity of the chlorohydrin. The chlorohydrin and epoxide show increased cytotoxicity compared to isohydroxypsorofebrin against a range of human tumor cell lines.

Glycosyl phosphates have emerged as useful glycosyl donors for stereoselective glycosidic bond formation in the synthe- sis of oligosaccharides. In this account, we describe some of our work in developing and further expanding this methodology, partic- ularly with the use of propane-1,3-diyl phosphate as the anomeric leaving group. We have studied the reactions of several glycosyl propane-1,3-diyl phosphates with or without C-2 participating groups with a range of glycosyl acceptors in the presence of trime- thylsilyl triflate in either stoichiometric or catalytic amount, and demonstrated their application in the synthesis of several disaccha- rides and also the synthesis of some trisaccharides.

ARN-3261 is an orally bioavailable small molecule inhibitor of the Salt Inducible Kinase 2 (SIK2, 11 nM) and SIK3 (19 nM). Three isoforms of SIK (SIKs) proteins have been reported: SIK1 (SNF1LK), SIK2 (QIK), and SIK3 (QSK). They are the Ser/Thr centrosome kinase family members required for bipolar mitotic spindle formation. The overexpression of SIK2 kinase in 30% of ovarian cancer specimens allows a novel, clinically important new method of treating ovarian cancer by blocking SIK2 kinase activity. In addition to a role in ovarian cancer, SIK2 and SIK3 are prevalent in several other tumor types, including breast, prostate diffuse large B-cell lymphoma, and melanoma cancers. Inhibition of SIK2 has been reported to cause centrosome splitting in interphase, while SIK2 depletion blocked centrosome separation in mitosis and sensitized ovarian cancers to paclitaxel in culture and in vivo Xenograft models. Depletion of SIK2 also delayed G1/S transition and reduced AKT phosphorylation. Higher levels of expression of SIK2 have been shown to be highly correlated with poor survival in patients with high-grade serous ovarian cancers. Using the homology structure of SIK2, fragment-based lead optimization strategies, and screening and structure-activity relationship efforts, we have discovered ARN-3261, a first-in-class novel, selective inhibitor of SIK2 that could prove useful in treating ovarian, endometrial, primary peritoneal, fallopian tube, and triple negative breast cancers. ARN-3261 specifically inhibited SIK2-expressed SKOv3 cells with an IC50 of 92 nM. ARN3261 was effective against ovarian, breast cancer cell lines alone and in combination with Paclitaxel and Cisplatin. ARN-3261 also inhibited ovarian tumor growth significantly at 70% in SKOv3 human ovarian cancer Xenografts in Ncr nu/nu mice in a dose dependent manner at 20, 40, 60, and 100 mg/kg orally. Moreover, ARN-3261 has exhibited excellent in vivo pharmacokinetic, pharmacodynamics, and correlative PK/PD and ADME characteristics. Preliminary in vitro and in vivo tumor up-take studies suggest that ARN-3261 blocks centrosome separation by inhibiting SIK2, thereby enhancing the sensitivity of Paclitaxel. Encouraged by these results, we initiated the IND enabling GLP-Toxicology and safety studies to bring ARN-3261 to the clinic for First-In-Human (FIH) Phase 1 trials. The non-clinical pharmacology data along with Phase 1 POC clinical trial plans will be presented. Citation Format: Hariprasad Vankayalapati, Venkatakrishnareddy Yerramreddy, Jinhua Zhou, Jeffrey A. Handler, Rajendra P. Appalaneni, Ramamohan R. Kancherla, Roy J. Wu, Hiroshi Takemori, Angelique Whitehurst, Amir Anthony Jazaeri, Robert L. Coleman, Zhen Lu, Robert C. Bast. Discovery of ARN-3261 as a potent, selective, orally available SIK2 inhibitor for treating ovarian, endometrial, primary peritoneal, fallopian tube, and triple negative breast cancers [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-296. doi:10.1158/1538-7445.AM2017-LB-296