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Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are standard of care in combination with endocrine therapy in advanced hormone receptor-positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Despite improved progression-free and overall survival, almost all patients develop CDK4/6 inhibitor resistance and experience disease progression on treatment. Aberrant activation of CDK2/cyclin E is a key resistance mechanism by which tumors can evade CDK4/6 blockade. Therefore, patients with HR+/HER2- breast cancer could benefit from treatment with a selective CDK2 inhibitor in combination with CDK4/6 inhibitors both in the resistant and first-line (1L) settings. BLU-222 is a novel, potent, and selective small-molecule inhibitor of CDK2 with favorable pharmacokinetic properties when administered orally, currently in early-stage clinical development. In pre-clinical studies using the MCF-7 xenograft model of HR+ CDK4/6-responsive breast cancer, treatment with BLU-222 combined with the CDK4/6 inhibitor ribociclib led to pronounced and durable tumor regression superior to ribociclib alone. In a derived palbociclib resistant MCF-7 xenograft model, ribociclib had no anti-tumor activity while BLU-222 led to a strong and durable anti-tumor response (83% tumor growth inhibition [TGI]) that was further improved when given in combination with ribociclib (110% TGI). To further explore the mechanism of aberrant CDK2 activation in CDK4/6 resistant, HR+ breast cancer, we engineered isogenic T47D cell lines to overexpress cyclin E1 (CCNE1) with or without p16, an endogenous inhibitor of CDK4/6 activity. In in vitro proliferation assays, co-expression of CCNE1 and p16 sensitized T47D cells to BLU-222 by approximately 10-fold compared to the parental control (110 nM vs 1078 nM, respectively). CDK4/6 inhibition with ribociclib had no anti-proliferative effect in the CCNE1 overexpressing cell lines regardless of p16 expression status. In vivo, treatment of the empty vector control T47D xenografts with ribociclib led to tumor stasis, while ribociclib in combination with BLU-222 led to tumor regression. T47D xenografts overexpressing both CCNE1 and p16 were resistant to ribociclib however CDK2 inhibition with BLU-222 single-agent treatment led to tumor regression. Finally, the activity of BLU-222 was evaluated in a patient-derived xenograft (PDX) model of CDK4/6 inhibitor-resistant HR+/HER2- breast cancer where the patient had progressed on 1L palbociclib/fulvestrant and 2L abemaciclib/fulvestrant therapy. In this PDX model, BLU-222 in combination with ribociclib led to tumor stasis, even in the absence of fulvestrant. In conclusion, these data support CDK2/cyclin E activation as a key vulnerability in CDK4/6 resistant, HR+/HER2- breast cancer and provide a rationale for the study of BLU-222 in patients with disease progression following CDK4/6 inhibitor treatment. Additionally, the improved durability of response when BLU-222 is combined with CDK4/6 inhibitors in the CDK4/6-naïve setting supports the combination of these agents as 1L treatment. BLU-222 is currently under investigation in VELA (NCT05252416), a phase 1/2, first-in-human trial for patients with cyclin E aberrant cancers and HR+/HER2- breast cancer. Citation Format: Victoria Brown, Nealia House, Phil Ramsden, Karen Ho, Hsin-Jung Wu, Erik Wilker, Jian Guo, Maxine Chen, Douglas Wilson, Neil Bifulco, Steve Wenglowsky, Yoon Jong Choi, Kerrie Faia. CDK2 inhibition with BLU-222 in combination with ribociclib demonstrates robust antitumor activity in pre-clinical models of CDK4/6 inhibitor-naïve and -resistant HR+/HER2- breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-07.

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic–lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2–M arrest and DNA damage. The VRK1–VRK2 synthetic–lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line–derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. Significance: A paralog synthetic–lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Supplementary Tables S1-S7

USP1 and PARP inhibition are synergistic in a BRCA1/2-dependent manner - related to Figure 4

CRISPR-UMI analysis and characterization of PCNA dynamics - related to Figure 3

Supplementary methods

I-138 inhibits USP1-UAF1 - related to Figure 1

I-138 activity in BRCA1/2 WT and mutant cells - related to Figure 2

CRISPR-UMI analysis of screens performed in NCI-H1693 cells - related to Figure 5

Supplementary Data from VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

Supplementary Figure from VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic–lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2–M arrest and DNA damage. The VRK1–VRK2 synthetic–lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line–derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.Significance:A paralog synthetic–lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

CRISPR Cas9-based screening is a powerful approach for identifying and characterizing novel drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacologic inhibition of USP1 leads to decreased DNA synthesis concomitant with S-phase–specific DNA damage. Genome-wide CRISPR-Cas9 screens identify RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition is associated with reduced PCNA protein levels. Ectopic expression of WT or ubiquitin-dead K164R PCNA reverses USP1 inhibitor sensitivity. Our results show, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to selected BRCA1/2 WT cancer cell lines enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant tumors. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to posttranslational modifications of PCNA. Taken together, USP1 inhibition may represent a novel therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.

Glioblastoma multiforme (GBM) is one of the most aggressive primary malignant brain tumors in adults. The median overall survival (OS) of GBM patients is poor (1-2 years) on standard of care therapies, which include surgery, radiotherapy, and the alkylating agent temozolomide. Therefore, developing new treatments for GBM is an urgent unmet medical need. TNG908 is a clinical stage MTA-cooperative PRMT5 inhibitor that inhibits PRMT5 activity selectively in MTAP-null cells leveraging a synthetic lethal interaction. Approximately 40% of GBM tumors are homozygous null for MTAP [1, 2]. In preclinical in vitro studies, the antiproliferation activity of TNG908 was 15 times more potent in MTAP-null cancer cell lines than in MTAP WT cells. TNG908 has high passive permeability and is not a substrate for P-glycoprotein nor Breast Cancer Resistant Protein efflux transporters, attributes which are favorable for crossing the blood-brain barrier. TNG908 demonstrated in vivo brain penetration in multiple preclinical studies, including non-human primates and mice, suggesting that TNG908 may fulfill the significant unmet need in the treatment of GBM. Pharmacodynamic activity of TNG908 to inhibit PRMT5 was demonstrated by decreased SDMA-modified protein levels in a dose-dependent manner in a GBM xenograft model. TNG908 demonstrated dose-dependent antitumor activity in multiple hyper- and hypomethylated GBM subcutaneous models, including cell lines and patient-derived xenograft models. Despite reduced Kpuu in rodents compared to primates, oral administration of TNG908 drove near tumor stasis and increased median survival by 3-fold in a highly aggressive murine GBM orthotopic model. In summary, TNG908 is a potent, brain-penetrant small molecule PRMT5 inhibitor that is selective for MTAP-null tumor cells with good drug-like properties and strong antitumor activity in preclinical models of GBM. As such, TNG908 may provide a novel treatment strategy for MTAP-deleted GBM patients. *MZ and AT contributed equally to the work. Reference: 1.Cerami et al., Cell, 2013. 2.Gao et al., Sci Signal, 2013 Citation Format: Minjie Zhang, Alice Tsai, Kevin M. Cottrell, Brian B. Haines, Erik Wilker, Heather DiBenedetto, Ron Weitzman, Alan Huang, Charles B. Davis, John P. Maxwell, Kimberly J. Briggs. TNG908, a brain-penetrant MTA-cooperative PRMT5 inhibitor, is efficacious in preclinical glioblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3452.

Synthetic lethality — a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone — can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interaction by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting Vaccinia-Related Kinase 1 (VRK1) in Vaccinia-Related Kinase 2 (VRK2)-methylated glioblastoma (GBM). VRK2 is silenced by promoter methylation in approximately two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-methylated GBM cell lines and patient-derived models was lethal and resulted in decreased activity of the downstream substrate Barrier to Autointegration Factor (BAF), a regulator of post-mitotic nuclear envelope formation. VRK1 knockdown, and thus reduced BAF activity, caused nuclear lobulation, blebbing and micronucleation, which subsequently resulted in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic VRK2 expression. Knockdown of VRK1 led to robust tumor growth inhibition in VRK2-methylated GBM xenografts. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

Synthetic lethality — a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone — can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interaction by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting Vaccinia-Related Kinase 1 (VRK1) in Vaccinia-Related Kinase 2 (VRK2)-methylated glioblastoma (GBM). VRK2 is silenced by promoter methylation in approximately two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells results in decreased activity of the downstream substrate Barrier to Autointegration Factor (BAF), a regulator of post-mitotic nuclear envelope formation. VRK1 knockdown, and thus reduced BAF activity, causes nuclear lobulation, blebbing and micronucleation, which subsequently results in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction is dependent on VRK1 kinase activity and is rescued by ectopic VRK2 expression. Knockdown of VRK1 leads to robust tumor growth inhibition in VRK2-methylated GBM xenografts. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia.

Purpose/Objective(s): Despite improvements in the treatment of advanced prostate cancer, there remains a major unmet need for further treatment options in the setting of metastatic castration-resistant prostate cancer (mCRPC). Recent evidence has pinpointed androgen receptor (AR) co-activators, such as p300/CREB binding protein (CBP), as potential therapeutic targets. Here, we describe the impact of a novel small molecule inhibitor of the p300/CBP bromodomain, FT-6876, in preclinical models of mCRPC. Materials/Methods: FT-6876 is a potent inhibitor of the bromodomains of CBP and p300 with high selectivity against other bromodomain-containing proteins (Caligiuri et al. Proceedings: AACR Annual Meeting 2020; Cancer Research. DOI: 10.1158/1538-7445.AM2020-3079). FT-6876 was tested, both alone and in combination with enzalutamide, in a panel of enzalutamide-sensitive and enzalutamide-resistant human prostate cancer cell lines, including LNCaP, LN95, and CS2. A Snail-inducible system, which confers enzalutamide resistance, in both LNCaP and LN95 lines, was also used. To test the effect of FT-6876 in inhibiting prostate cancer growth in vitro, 2D colony forming assays and 3D spheroid assays were performed. RT-qPCR and multi-omics analysis were performed to assess changes in gene and protein expression. To test the effects of FT-6876 in physiologic settings, an ex vivo murine pulmonary metastasis assay (PuMA) as well as a patient-derived xenograft (PDX) model were used. Results: FT-6876 treatment resulted in concentration-dependent growth inhibition of all tested enzalutamide-sensitive and -resistant cell lines. Treated cells showed decreased expression of several key prostate cancer genes, including KLK3 (PSA), TMPRSS2, and MYC. A multi-omics approach using proteomics, acetylomics, and RNA-Seq illuminated the global landscape of alterations in epigenetic and downstream signaling pathways induced by FT-6876. Changes were observed in both enzalutamide-sensitive and -resistant models. Finally, FT-6876 reduced metastatic growth in the PuMA model and induced tumor stasis in a patient-derived xenograft model of prostate cancer resistant to enzalutamide. Conclusion: Overall, we demonstrate that inhibiting the p300/CBP axis with the novel bromodomain inhibitor FT-6876 could be an effective therapeutic approach for both enzalutamide-sensitive and enzalutamide-resistant prostate cancer models. The safety and tolerability of FT-7051, an oral p300/CBP inhibitor related to FT-6876, is currently being evaluated in the Courage Study (NCT04575766), a first-in-human phase 1 study in men with mCRPC. Citation Format: Emily L. Chen, Nathan Hawkey, Beatrice C. Thomas, Kathryn E. Ware, Daniella Runyambo, Maureen Caligiuri, Erik Wilker, Erik J. Soderblom, M. Arthur Moseley III, Sylvie M. Guichard, Andrew J. Armstrong, Jason A. Somarelli. Targeting the p300/CBP epigenetic pathway to overcome hormone therapy resistance in advanced prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P204.

PRMT5 is a type II arginine methyltransferase that regulates essential cellular functions via symmetric dimethylation of target proteins involved in spliceosome regulation, cell cycle progression, apoptosis, the DNA-damage response, and other functions. PRMT5 dependence in cells with MTAP deletions is a strong and prevalent synthetic lethal interaction. Due to their mechanisms of action, existing clinical PRMT5 inhibitors do not recapitulate the selectivity for MTAPnull cells demonstrated by genetic perturbation. Given that MTAP deletion occurs in approximately 10-15% of all human cancer[i],[ii],[iii], a molecule that selectively kills MTAPnull cancer cells provides an important opportunity to deliver a targeted treatment to a significant patient population. We have discovered small molecules that exhibit MTA-cooperative PRMT5 binding and selectively kill MTAPnull cancer cells. Striking MTAP-dependent viability effects are demonstrated in MTAP-isogenic cell lines representing multiple lineages, and in a multi-lineage cell line panel comprised of 200 cancer cell lines. Furthermore, oral administration of an MTAPnull-selective PRMT5 inhibitor demonstrates dose-dependent antitumor activity and strong regressions across multiple histologies in both cancer cell line and patient-derived xenografts. These data suggest the therapeutic potential of MTAPnull-selective PRMT5 inhibitors in MTAP-deleted cancers. [i] Cerami et al., 2012 [ii] Gao et al., 2013 [iii] Lee et al., 2014 Citation Format: Kimberly J. Briggs, Kevin M. Cottrell, Matthew R. Tonini, Erik Wilker, Lina Gu, Charles B. Davis, Doug Whittington, Deepali Gotur, Haris Jahic, Matthew J. Goldstein, Alan Huang, John P. Maxwell. MTAPnull-selective PRMT5 inhibitors drive regressions in MTAP-deleted xenograft models across histologies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P214.

Synthetic lethality — the genetic interaction that causes cell death when two genetic deficiencies co-occur but cell survival when each deficiency occurs alone — can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interactions by virtue of their redundant function in the cell. Here we demonstrate a paralog synthetic lethality by targeting VRK1 in VRK2-methylated glioblastoma multiforme (GBM). VRK2 is silenced by methylation in about two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells leads to cell death via cell cycle arrest in G2M and subsequent DNA damage. The lethality is dependent on the kinase activity of VRK1 and is rescued by ectopic VRK2 expression. Knockdown of VRK1 leads to robust tumor growth inhibition in VRK2-methylated glioblastoma xenografts in vivo. These results suggest that inhibiting VRK1 kinase activity could be a viable cancer therapeutic in VRK2-methylated glioblastoma. Citation Format: Julie Shields, Samuel R. Meier, Justin Engel, Madhavi Bandi, Maria L. Dam Ferdinez, Wenhai Zhang, Shan-chuan Zhao, Minjie Zhang, Ashley Choi, Yi Yu, Xuewen Pan, Brian McMillan, Brett Williams, Robert Tjin Tham Sjin, Douglas Whittington, Erik Wilker, Alan Huang, Fang Li, Natasha Emmanuel. VRK1 is a novel synthetic lethal target in VRK2-methylated glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P182.

Triple negative breast cancer (TNBC) remains a high unmet medical need, commonly treated with chemotherapies. Biomarker-driven treatment strategies have led to the recent approvals of both PARP inhibitors and an immune checkpoint inhibitor for a subset of women with TNBC. Androgen receptor (AR) is expressed in a 20-30% of patients with TNBC. Preclinical studies indicate that AR in this context can substitute for estrogen receptor (ER) to drive transcription of ER target genes, supporting TNBC tumor proliferation and survival. AR-driven transcription is dependent upon multiple essential cofactors, among which are the structurally related, bromodomain (BRD) and lysine acetyl transferase containing proteins, CBP and p300. Here we report the discovery of FT-6876, a potent and selective small molecule bromodomain inhibitor of CBP/p300. Our hypothesis is that modulation of CBP/p300 with FT-6876 will alter chromatin compaction and, when in the vicinity of AR responsive genes, selectively reduce AR transcriptional activity at these loci. This should translate to growth inhibition in vitro and in vivo in AR+ breast cancer models. Consistent with this hypothesis, a rapid reduction in H3K27Ac was observed in an AR+ breast cancer cell line following treatment with FT-6876. Removal of the compound led to the restoration of this mark to baseline levels reflecting the dynamic state of histone modification in cells. High content analysis revealed that the reduction in H3K27Ac occurred in the majority of the cells. In order to have a greater understanding of the dynamic and selective impact of FT-6876 on AR/ER transcriptional activity, we used precision run-on (PRO) seq to quantify nascent RNA and infer transcription factor modulation genome-wide. GSEA analysis of nascent RNA showed a high enrichment for AR and ER pathway genes upon exposure with FT-6876. Using ChIPseq, we confirmed that the reduction of H3K27Ac at specific promoter sites was concurrent with a decrease in CBP/p300 on the chromatin and reduction in nascent RNA and eRNA. FT-6876 reduced proliferation in a time-dependent manner with optimal growth inhibition observed after 10 days drug exposure. This was maintained even after removal of the drug for several days. Across a panel of breast cancer cell lines, there was a good correlation between growth inhibition and AR expression. In vivo, FT-6876 induced tumor stasis at well tolerated doses in AR+ breast cancer models. This was associated with a reduction in H3K27Ac, AR and ER target gene modulation, and reduction in Ki67. FT-6876 is a promising new CBP/p300 bromodomain inhibitor demonstrating efficacy in preclinical models of AR+ breast cancer. Citation Format: Maureen Caligiuri, Grace L. Williams, Jennifer Castro, Linda Battalagine, Kristina Muskiewicz, Erik Wilker, Lili Yao, Shawn Schiller, Angela Toms, Ping Li, Eneida Pardo, Bradford Graves, Kenneth W. Wood, Sylvie M. Guichard. FT-6876, a potent and selective inhibitor of CBP/p300 with antitumor activity in AR-positive 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 3079.

The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.

Isoelectric focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly(vinyl alcohol) (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370 V/cm), efficient cooling maintains solution temperature inside the separation channel controllably in the range 2-25 degrees C. Protein samples with a dynamic concentration range from microg/mL to mg/mL can be loaded into the microdevice at a flow rate of 1 mL/h and residence time of approximately 12 min. By using a protein complex of nine proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device-to-device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10-20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The microdevice has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches.

Akt is a serine/threonine kinase involved in a variety of cellular responses, including cell proliferation and cell survival. Recent studies from our laboratory suggest that Akt signaling may play an important role in skin tumor promotion. To explore this premise, we examined epidermal Akt activation and signaling in response to chemically diverse skin tumor promoters. Mice received single or multiple applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, or chrysarobin. All three tumor promoters were able to activate epidermal Akt as early as 1 h after treatment. Activation of Akt following tumor promoter treatment led to enhanced downstream signaling, including hyperphosphorylation of glycogen synthase kinase-3β and Bad. Structure activity studies with phorbol ester analogues revealed that the magnitude of activation paralleled tumor-promoting activity. In cultured primary keratinocytes, TPA treatment also led to activation of Akt. Activation of the epidermal growth factor receptor (EGFR) seemed to underlie the ability of TPA to activate Akt as both PD153035, an inhibitor of EGFR, and GW2974, a dual-specific inhibitor of both EGFR and erbB2, were able to effectively reduce TPA-induced Akt phosphorylation as well as TPA-stimulated EGFR and erbB2 tyrosine phosphorylation in a dose-dependent manner. Furthermore, inhibition of protein kinase C (PKC) activity blocked TPA-stimulated heparin-binding EGF production and EGFR transactivation. Inhibition of PKC also led to a decreased association of Akt with the PP2A catalytic subunit, leading to increased Akt phosphorylation. However, combination of EGFR inhibitor and PKC inhibitor completely abrogated TPA-induced activation of Akt. Collectively, the current results support the hypothesis that elevated Akt activity and subsequent activation of downstream signaling pathways contribute significantly to skin tumor promotion. In addition, signaling through the EGFR via EGFR homodimers or EGFR/erbB2 heterodimers may be the primary event leading to Akt activation during tumor promotion in mouse skin. (Mol Cancer Res 2007;5(12):1342–52)

Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate Rac1 signaling by inhibiting its downstream effector kinases, the p21-activated kinases (Pak). Given the implication of Paks in tumorigenesis, it is plausible that merlin's tumor suppressive function might be mediated, at least in part, via inhibition of the Paks. We present data indicating this is indeed the case. First, analysis of primary schwannoma samples derived from NF2 patients showed that in a significant fraction of the tumors, the activity of Pak1 was highly elevated. Second, we used shRNAs to knockdown Pak1, 2, and 3 in NIH3T3 cells expressing a dominant-negative form of merlin, NF2BBA (NIH3T3/NF2BBA), and find that simultaneous knockdown of Pak1-3 in these cells significantly reduced their growth rates in vitro and inhibited their ability to form tumors in vivo. Finally, while attempting to silence Pak1 in rat schwannoma cells, we found that these cells were unable to tolerate long-term Pak1 inhibition and rapidly moved to restore Pak1 levels by shutting down Pak1 shRNA expression through a methylation-dependent mechanism. These data suggest that inhibiting Pak could be a beneficial approach for the development of therapeutics toward NF2. In addition, the finding that the shRNA-mediated Pak1 suppression was silenced rapidly by methylation raises questions about the future application of such technologies for the treatment of diseases such as cancer. [Cancer Res 2008;68(19):7932–7]

The PI3K pathway is involved in the regulation of cell growth, proliferation, metabolism and other functions. Aberrant signaling (PTEN loss of function, PIK3CA mutation, Akt amplification, etc.) from the PI3K pathway is observed in >50% of all tumors. Clinical evidence suggests that inhibiting the PI3K pathway is beneficial for the treatment of solid tumors and tumors of the hematopoietic system. Inhibition of mTOR via rapalogs has been shown to block a negative feedback loop, thereby leading to the activation of Akt. The activation of this Akt feedback loop has been suggested to potentially compromise the clinical efficacy of selective mTORC1 inhibitors such as temsirolimus and everolimus. Dual p70S6K/Akt inhibition may promote improved pathway inhibition and also block the negative consequences of Akt activation through the negative feedback loop. MSC2363318A is a highly selective, potent, adenosine triphosphate (ATP) competitive inhibitor of p70S6K, Akt1, and Akt3. In a cellular context, inhibition of p70S6K leads to potent inhibition of ribosomal protein S6 phosphorylation, while inhibition of Akt activity blocks the negative effects of a compensatory feedback loop. In addition, MSC2363318A exhibits potent anti-proliferative activity against many solid tumor cell lines in vitro, especially those with PI3K pathway genomic alterations. Further, MSC2363318A can also cross the blood-brain barrier (via pre-clinical studies in mice, rat, and dog), a unique characteristic that would allow for treating not only malignancies that are driven by PI3K pathway genomic alterations, but also indications with a high incidence of CNS metastases and primary malignancies of the central nervous system. Patient Derived Xenograft (PDX) models from breast cancers with a high prevalence of PI3K pathway genomic alterations; including, triple negative breast cancer and Her2+ breast cancer were evaluated. In addition, combinations with standard of care agents were then evaluated in these breast cancer PDX models. Results from these studies were correlated with PI3K pathway genomic modifications, and will be used to guide subsequent clinical studies. Citation Format: Bayard R. Huck, H Tian, Sakeena Syed, Jing Lin, Jianguo Ma, Anderson Clark, Remiguisz Kaleta, Andreas Machl, Erik Wilker, Marc Lecomte. Evaluation of p70S6K/Akt inhibitor MSC2363318A in patient derived xenograft (PDX) models of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4516. doi:10.1158/1538-7445.AM2014-4516

The PI3K pathway is involved in the regulation of cell growth, proliferation, metabolism and other functions. Aberrant signaling (PTEN loss of function, PIK3CA mutation, Akt amplification, etc.) from the PI3K pathway is observed in >50% of all tumors. Clinical evidence suggests that inhibiting the PI3K pathway is beneficial for the treatment of solid tumors and tumors of the hematopoietic system. Inhibition of p70S6K via rapalogs has been shown to block a negative feedback loop, thereby leading to the activation of Akt. The activation of this Akt feedback loop has been suggested to potentially compromise the clinical efficacy of selective mTORC1 inhibitors such as temsirolimus and everolimus. Dual p70S6K/Akt inhibition may promote improved pathway inhibition and also block the negative consequences of Akt activation through the negative feedback loop. MSC2363318A is a highly kinase-selective, potent, adenosine triphosphate (ATP) competitive inhibitor of p70S6K, Akt1, and Akt3. In a cellular context, inhibition of p70S6K leads to potent inhibition of ribosomal protein S6 phosphorylation, while inhibition of Akt activity blocks the negative effects of a compensatory feedback loop. In addition, MSC2363318A exhibits potent anti-proliferative activity against many solid tumor cell lines in vitro, especially those with PI3K pathway genomic alterations. Further, MSC2363318A can also cross the blood-brain barrier, a unique characteristic that would allow for treating not only primary malignancies that are driven by PI3K pathway genomic alterations, but also indications with a high incidence of CNS metastases and primary malignancies of the central nervous system. Oral treatment of mice with MSC2363318A resulted in significant inhibition of tumor growth in several subcutaneous human cancer xenograft models representing breast, pancreatic, glioblastoma and ovarian cancers. Of note, a breast cancer model possessing a PTEN loss of function showed tumor regression upon treatment with MSC2363318A. In addition, MSC2363318A exhibited increased exposure in tumors as compared to plasma, resulting in sustained inhibition of S6K phosphorylation for up to 24 hours. Key preclinical activities are being completed and first in human clinical studies are scheduled to be initiated in 2013. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A162. Citation Format: Bayard R. Huck, Andreas Machl, Erik Wilker, Hui Tian, Sakeena Syed, Jing Lin, Jianguo Ma, Anderson Clark, Roseann Waterhouse, Remiguisz Kaleta, Alina Micu, Lynne Soughley, Long Li, Karin Groll, Sonja Kroesser, Frank Beier, Ursula Hering, Marc Lecomte, Katrin Wichert, Mauro D'Antonio, Paolo Di Eugenio. Identification of brain penetrant p70S6K/Akt inhibitor MSC2363318A. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A162.