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Venetoclax combination therapies are becoming the standard of care in acute myeloid leukemia (AML). However, the therapeutic benefit of these drugs in older/unfit patients is limited to only a few months, highlighting the need for more effective therapies. Protein phosphatase 2A (PP2A) is a tumor suppressor phosphatase with pleiotropic functions that becomes inactivated in ∼70% of AML cases. PP2A promotes cancer cell death by modulating the phosphorylation state in a variety of proteins along the mitochondrial apoptotic pathway. We therefore hypothesized that pharmacological PP2A reactivation could increase BCL2 dependency in AML cells and, thus, potentiate venetoclax–induced cell death. Here, by using 3 structurally distinct PP2A-activating drugs, we show that PP2A reactivation synergistically enhances venetoclax activity in AML cell lines, primary cells, and xenograft models. Through the use of gene editing tools and pharmacological approaches, we demonstrate that the observed therapeutic synergy relies on PP2A complexes containing the B56α regulatory subunit, of which expression dictates response to the combination therapy. Mechanistically, PP2A reactivation enhances venetoclax-driven apoptosis through simultaneous inhibition of antiapoptotic BCL2 and extracellular signal-regulated kinase signaling, with the latter decreasing MCL1 protein stability. Finally, PP2A targeting increases the efficacy of the clinically approved venetoclax and azacitidine combination in vitro, in primary cells, and in an AML patient-derived xenograft model. These preclinical results provide a scientific rationale for testing PP2A-activating drugs with venetoclax combinations in AML.

High-grade serous carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP inhibitors (PARPi) have become the mainstay of HGSC-targeted therapy, given that these tumors are driven by a high degree of genomic instability (GI) and homologous recombination (HR) defects. Nonetheless, approximately 30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and an increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a small-molecule activator of protein phosphatase 2A (PP2A; SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in patient-derived HGSC cells and xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for cellular transformation (B56α, B56γ, and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061–induced stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and HR-deficient HGSC cells and PDX models. Our studies identify PP2A as a novel regulator of HR and indicate PP2A modulators as a therapeutic therapy for HGSC. In summary, our findings further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 presents benefits in overcoming PARPi resistance driven by BRCA1/2 mutation reversions.

Small-cell lung cancer (SCLC) is the deadliest form of lung cancer but lacks targeted therapies.We studied the effect of the natural product mistletoe lectin (ML) in pre-clinical models of SCLC, focusing on cell lines with amplification of the myc family oncogenes C-myc and N-myc.We found that ML treatment inhibits growth of SCLC cell lines in culture and induces apoptosis. ML treatment also decreases the expression of the amplified myc proteins. Over-expression of either C-myc or N-myc results in enhanced SCLC cell sensitivity to ML. In a mouse xenograft model of SCLC, treatment with ML results in decreased tumor growth over 4 weeks with evidence of increased apoptosis in tumors from treated animals.Overall, our results demonstrate that ML exhibits therapeutic potential in SCLC, that is at least partially dependent on myc protein expression.

Fig. S1

High-grade serous carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP inhibitors (PARPi) have become the mainstay of HGSC-targeted therapy, given that these tumors are driven by a high degree of genomic instability (GI) and homologous recombination (HR) defects. Nonetheless, approximately 30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and an increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a small-molecule activator of protein phosphatase 2A (PP2A; SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in patient-derived HGSC cells and xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for cellular transformation (B56α, B56γ, and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061–induced stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and HR-deficient HGSC cells and PDX models. Our studies identify PP2A as a novel regulator of HR and indicate PP2A modulators as a therapeutic therapy for HGSC. In summary, our findings further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 presents benefits in overcoming PARPi resistance driven by BRCA1/2 mutation reversions.

Table S1

Legends for Supplementary Tables 1,2 and Figures S1-S3

Erlotinib is a tyrosine kinase inhibitor approved for the treatment of patients with advanced non–small cell lung cancer (NSCLC). In these patients, erlotinib prolongs survival but its benefit remains modest because many tumors express wild-type (wt) EGFR or develop a second-site EGFR mutation. To test drug combinations that could improve the efficacy of erlotinib, we combined erlotinib with quinacrine, which inhibits the FACT (facilitates chromatin transcription) complex that is required for NF-κB transcriptional activity. In A549 (wtEGFR), H1975 (EGFR-L858R/T790M), and H1993 (MET amplification) NSCLC cells, this drug combination was highly synergistic, as quantified by Chou–Talalay combination indices, and slowed xenograft tumor growth. At a sub-IC50 but more clinically attainable concentration of erlotinib, quinacrine, alone or in combination with erlotinib, significantly inhibited colony formation and induced cell-cycle arrest and apoptosis. Quinacrine decreased the level of active FACT subunit SSRP1 and suppressed NF-κB–dependent luciferase activity. Knockdown of SSRP1 decreased cell growth and sensitized cells to erlotinib. Moreover, transcriptomic profiling showed that quinacrine or combination treatment significantly affected cell-cycle–related genes that contain binding sites for transcription factors that regulate SSRP1 target genes. As potential biomarkers of drug combination efficacy, we identified genes that were more strongly suppressed by the combination than by single treatment, and whose increased expression predicted poorer survival in patients with lung adenocarcinoma. This preclinical study shows that quinacrine overcomes erlotinib resistance by inhibiting FACT and cell-cycle progression, and supports a clinical trial testing erlotinib alone versus this combination in advanced NSCLC. Mol Cancer Ther; 13(9); 2203–14. ©2014 AACR.

Karyotype analysis for Spermatogonia at passage 16, before transfection with XTNs demonstrating a normal male 40, XY karyotype.

ANOVA with multiple comparisons and Tukey's post-hoc test

Supplementary Data figures and tables Fig. S1. Genomic characterization of CR cells. Fig. S2. Karyotype analysis of CR cells. Fig. S3. Drug sensitivity profiles of CR cells. Fig. S4. Population doubling times of Parental and nab-paclitaxel resistant cells. Fig. S5. Nab-paclitaxel sensitivity of CR spheroids. Fig. S6. Dependency of CR cells on KRAS. Fig. S7. CR-based PDX tumors in mice. Fig. S8. Reverse Phase Proteomic analysis of Parental and nab-paclitaxel resistant cells. Fig. S9. Modulation of c-Myc and the effects on nab-paclitaxel sensitivity. Fig. S10. SMAP2 Responses. Table S1. Patient data Table S2. Gene mutations associated with nab-paclitaxel resistance

Supplementary Table S1. AML patients. Supplementary Table S2. RT-qPCR primers. Supplementary Table S3. IC50 concentrations (nM) of FLT3 inhibitors, PP2A activators and GSK-3beta inhibitors. Supplementary Figure S1. Concurrent PP2A-activating drug treatment increases cytotoxicity of FLT3 inhibitors in Ba/F3-ITD cells with FLT3-ITD and in an MV4-11 orthotopic mouse model. Supplementary Figure S2. Chou-Talalay analysis of FLT3-WT cell lines. Supplementary Figure S3. Images of all mice in the in vivo experiment. Supplementary Figure S4. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor does not induce apoptosis in wild-type FLT3 AML, AML complete remission (CR) or AML complete remission with incomplete platelet recovery (CRp) marrows. Supplementary Figure S5. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor downregulates c-Myc and Pim-1 protein expression in cells with FLT3-ITD. Supplementary Figure S6. p-c-Myc (T58) and p-c-Myc (S62) expression in Ba/F3-ITD and MV4-11 cells treated with gilteritinib and/or FTY720. Supplementary Figure S7. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor produces variable changes in c-Myc and Pim-1 mRNA expression in cells with FLT3-ITD. Supplementary Figure S8. c-Myc and Pim-1 expression in FLT3-WT AML blasts treated with gilteritinib and/or FTY720. Supplementary Figure S9. Proteasome inhibition inhibits downregulation of c-Myc and Pim-1 expression by concurrent PP2A-activating drug and FLT3 inhibitor treatment. Supplementary Figure S10. Concurrent PP2A-activating drug and FLT3 inhibitor treatment increases c-Myc and Pim-1 protein turnover in cells with FLT3-ITD. Supplementary Figure S11. Concurrent PP2A activator and FLT3 inhibitor treatment downregulates p-ERK later than p-AKT in cells with FLT3-ITD. Supplementary Figure S12. The GSK-3beta inhibitor TWS119 prevents c-Myc and Pim-1 downregulation and apoptosis induction by PP2A-activating drug and FLT3 inhibitor combination in Ba/F3-ITD cells.

Supplementary Figures S1-S12, Supplementary Table S1-3, supplementary methods and reference.

Fms-like tyrosine-like kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients and is associated with short disease-free survival. FLT3 inhibitor efficacy is limited and transient but may be enhanced by multitargeting of FLT3-ITD signaling pathways. FLT3-ITD drives both STAT5-dependent transcription of oncogenic Pim-1 kinase and inactivation of the tumor-suppressor protein phosphatase 2A (PP2A), and FLT3-ITD, Pim-1, and PP2A all regulate the c-Myc oncogene. We studied mechanisms of action of cotreatment of FLT3-ITD–expressing cells with FLT3 inhibitors and PP2A-activating drugs (PADs), which are in development. PADs, including FTY720 and DT-061, enhanced FLT3 inhibitor growth suppression and apoptosis induction in FLT3-ITD–expressing cell lines and primary AML cells in vitro and MV4-11 growth suppression in vivo. PAD and FLT3 inhibitor cotreatment independently downregulated c-Myc and Pim-1 protein through enhanced proteasomal degradation. c-Myc and Pim-1 downregulation was preceded by AKT inactivation, did not occur in cells expressing myristoylated (constitutively active) AKT1, and could be induced by AKT inhibition. AKT inactivation resulted in activation of GSK-3β, and GSK-3β inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3β activation increased c-Myc proteasomal degradation through c-Myc phosphorylation on T58; infection with c-Myc with T58A substitution, preventing phosphorylation, blocked downregulation of c-Myc by PAD and FLT3 inhibitor cotreatment. GSK-3β also phosphorylated Pim-1L/Pim-1S on S95/S4. Thus, PADs enhance efficacy of FLT3 inhibitors in FLT3-ITD–expressing cells through a novel mechanism involving AKT inhibition–dependent GSK-3β–mediated increased c-Myc and Pim-1 proteasomal degradation.

Metastatic castration-resistant prostate cancer (CRPC) is a fatal disease, primarily resulting from the transcriptional addiction driven by androgen receptor (AR). First-line CRPC treatments typically target AR signaling, but are rapidly bypassed, resulting in only a modest survival benefit with antiandrogens. Therapeutic approaches that more effectively block the AR-transcriptional axis are urgently needed. Here, we investigated the molecular mechanism underlying the association between the transcriptional coactivator MED1 and AR as a vulnerability in AR-driven CRPC. MED1 undergoes CDK7-dependent phosphorylation at T1457 and physically engages AR at superenhancer sites, and is essential for AR-mediated transcription. In addition, a CDK7-specific inhibitor, THZ1, blunts AR-dependent neoplastic growth by blocking AR/MED1 corecruitment genome-wide, as well as reverses the hyperphosphorylated MED1-associated enzalutamide-resistant phenotype. In vivo, THZ1 induces tumor regression of AR-amplified human CRPC in a xenograft mouse model. Together, we demonstrate that CDK7 inhibition selectively targets MED1-mediated, AR-dependent oncogenic transcriptional amplification, thus representing a potential new approach for the treatment of CRPC.Significance:Potent inhibition of AR signaling is critical to treat CRPC. This study uncovers a driver role for CDK7 in regulating AR-mediated transcription through phosphorylation of MED1, thus revealing a therapeutically targetable potential vulnerability in AR-addicted CRPC.See related commentary by Russo et al., p. 1490.This article is highlighted in the In This Issue feature, p. 1469

Results of Kinase Inhibitor Screen including compound names, IC50s, and fold change compared to vehicle.

STR DNA Profiling for Cell Line Authentication

Supplemental data for the classical and enhanced sampling molecular dynamics simulations

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC.Significance:A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.

Supplementary Materials and Methods.

Viability assays, Soft agar, Immunoprecipitation, Kinase inhibitor screen, and Patient outcome data

Somatic mutation of the protein phosphatase 2A (PP2A) Aα-subunit gene PPP2R1A is highly prevalent in high-grade endometrial carcinoma. The structural, molecular, and biological basis by which the most recurrent endometrial carcinoma–specific mutation site P179 facilitates features of endometrial carcinoma malignancy has yet to be fully determined. Here, we used a series of structural, biochemical, and biological approaches to investigate the impact of the P179R missense mutation on PP2A function. Enhanced sampling molecular dynamics simulations showed that arginine-to-proline substitution at the P179 residue changes the protein's stable conformation profile. A crystal structure of the tumor-derived PP2A mutant revealed marked changes in A-subunit conformation. Binding to the PP2A catalytic subunit was significantly impaired, disrupting holoenzyme formation and enzymatic activity. Cancer cells were dependent on PP2A disruption for sustained tumorigenic potential, and restoration of wild-type Aα in a patient-derived P179R-mutant cell line restored enzyme function and significantly attenuated tumorigenesis and metastasis in vivo. Furthermore, small molecule–mediated therapeutic reactivation of PP2A significantly inhibited tumorigenicity in vivo. These outcomes implicate PP2A functional inactivation as a critical component of high-grade endometrial carcinoma disease pathogenesis. Moreover, they highlight PP2A reactivation as a potential therapeutic strategy for patients who harbor P179R PPP2R1A mutations.Significance:This study characterizes a highly recurrent, disease-specific PP2A PPP2R1A mutation as a driver of endometrial carcinoma and a target for novel therapeutic development.See related commentary by Haines and Huang, p. 4009

Results of the Phosphokinase Array with phosphorylation sites, fold change relative to vehicle, and Kmeans clustering.

Supplemental Figures S1-S9. S1: Kinase inhibitor screen and response of OP449 and INK128. S2: CI values for DT1154 combined with INK128 and viability curves. S3: Kmeans clusters and signaling in response to PP2A activators and INK128. S4: Oncoprint and survival curves of patients with alterations in the MYC/mTOR pathway. S5: pT58 MYC expression levels in PDA cells treated with DT1154 and INK128. S6: Signaling with B56a knockdown or T58A MYC overexpression in response to DT1154 and INK128 treatment. S7: Signaling showing decreased expression of MYC increases the efficacy of INK128. S8: Treatment of KPC and KPCM/+ cell lines with BEZ235, Dasatinib, and Sunitinib. S9: Endpoint analysis of long- and short-term in vivo treatment with DT1154 and INK128.

Antibody information and primer sequences.

Supplementary Figure from Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma

Supplementary Figure Legends and Methods

Supplemental Figure 1. Comparison of the anti-proliferative activity of SMAP across a panel of cell lines. Supplemental Figure 2. The chemical structure of SMAPs utilized in the studies detailed in the manuscript. Supplemental Figure 3. The effects of SMAPs on cell proliferation and cell survival. Supplemental Figure 4. Control studies for co-immunoprecipitation experiments. Supplemental Figure 5. Activity of SMAPs in vivo with the correlation of anti-tumor activity with SMAP-2 exposure in serum of treated mice.

There is currently a lack of precise predictive biomarkers for patient selection in clinical trials of inhibitors targeting replication stress (RS) response proteins ATR and CHK1. The objective of this study was to identify novel predictive biomarkers for the response to these agents in treating non–small cell lung cancer (NSCLC). A genome-wide loss-of-function screen revealed that tumor suppressor PPP2R2A, a B regulatory subunit of protein phosphatase 2 (PP2A), determines sensitivity to CHK1 inhibition. A synthetic lethal interaction between PPP2R2A deficiency and ATR or CHK1 inhibition was observed in NSCLC in vitro and in vivo and was independent of p53 status. ATR and CHK1 inhibition resulted in significantly increased levels of RS and altered replication dynamics, particularly in PPP2R2A-deficient NSCLC cells. Mechanistically, PPP2R2A negatively regulated translation of oncogene c-Myc protein. c-Myc activity was required for PPP2R2A deficiency-induced alterations of replication initiation/RS and sensitivity to ATR/CHK1 inhibitors. We conclude that PPP2R2A deficiency elevates RS by upregulating c-Myc activity, rendering cells reliant on the ATR/CHK1 axis for survival. Our studies show a novel synthetic lethal interaction and identify PPP2R2A as a potential new predictive biomarker for patient stratification in the clinical use of ATR and CHK1 inhibitors.Significance:This study reveals new approaches to specifically target PPP2R2A-deficient lung cancer cells and provides a novel biomarker that will significantly improve treatment outcome with ATR and CHK1 inhibitors.

Basal-like breast cancers (BLBC) are characterized by defects in homologous recombination (HR), deficient mitotic checkpoint, and high-proliferation activity. Here, we discover CIP2A as a candidate driver of BLBC. CIP2A was essential for DNA damage–induced initiation of mouse BLBC-like mammary tumors and for survival of HR–defective BLBC cells. CIP2A was dispensable for normal mammary gland development and for unperturbed mitosis, but selectively essential for mitotic progression of DNA damaged cells. A direct interaction between CIP2A and a DNA repair scaffold protein TopBP1 was identified, and CIP2A inhibition resulted in enhanced DNA damage–induced TopBP1 and RAD51 recruitment to chromatin in mammary epithelial cells. In addition to its role in tumor initiation, and survival of BRCA-deficient cells, CIP2A also drove proliferative MYC and E2F1 signaling in basal-like triple-negative breast cancer (BL-TNBC) cells. Clinically, high CIP2A expression was associated with poor patient prognosis in BL-TNBCs but not in other breast cancer subtypes. Small-molecule reactivators of PP2A (SMAP) inhibited CIP2A transcription, phenocopied the CIP2A-deficient DNA damage response (DDR), and inhibited growth of patient-derived BLBC xenograft. In summary, these results demonstrate that CIP2A directly interacts with TopBP1 and coordinates DNA damage–induced mitotic checkpoint and proliferation, thereby driving BLBC initiation and progression. SMAPs could serve as a surrogate therapeutic strategy to inhibit the oncogenic activity of CIP2A in BLBCs.Significance:These results identify CIP2A as a nongenetic driver and therapeutic target in basal-like breast cancer that regulates DNA damage–induced G2–M checkpoint and proliferative signaling.

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065–80. ©2018 AACR.

Table S1. KSEA Analysis and the Mean FC Method in LNCaP treated with SMAP. Table S2. ANOVA with multiple comparisons and Dunnett''s post-hoc test for SMAP treated LNCaP colony formation assay. Table S3. ANOVA with multiple comparisons and Dunnett''s post-hoc test for SMAP treated 22Rv1 colony formation assay. Table S4. ANOVA with multiple comparisons and Dunnett''s post-hoc test for Annexin V staining of LNCap and 22Rv1 cells. Table S5. PPI of the phosphoproteomics dataset. Table S6. Kinase Substrate Database for LNCaP treated with SMAP. Table S7. ANOVA with multiple comparisons and Tukey''s post-hoc test for SMAP treatment in LNCaP cells phosphorylated to total AR ratio protein. Table S8. ANOVA for qRT-PCR RNA for AR targets in LNCaP and 22Rv1 cells in presence of SMAPs. Table S9. ANOVA with multiple comparisons and Dunnett''s post-hoc test for LNCaP and 22Rv1 cells treated with SMAP, bortezomib, and combination. Table S10. ANOVA with multiple comparisons and Tukey''s post-hoc test for relative AR protein expression in LNCaP- Small T stably expressing lines treated with SMAP. Table S11. ANOVA with multiple comparisons and Tukey''s post-hoc test for relative AR protein expression in LNCaP cells in FBS or CSS in presence or absence of R1881. Table S12. ANOVA for qRT-PCR RNA AR targets statistical analysis for LNCaP cells in FBS or CSS in presence or absence of R1881. Table S13. ANOVA with multiple comparisons and Dunnett''s post-hoc test for LnCaP/AR xenograft treatment study: vehicle control, SMAP 400mg/kg, SMAP 100mg/kg and MDV3100 100mg/kg. Table S14. ANOVA with multiple comparisons and Dunnett''s post-hoc test for castrated LNCaP/AR tumor volumes for individual treatments groups: Vehicle control, SMAP-2 100mg/kg and SMAP-2 30mg/kg. Table S15. ANOVA with multiple comparisons and Dunnett''s post-hoc test for TUNEL positive staining quantification from castrated LNCaP/AR tumors from individual treatments groups: Vehicle control, SMAP-2 30mg/kg and SMAP-2 100mg/kg. Table S16. ANOVA with multiple comparisons and Dunnett''s post-hoc test for PCNA positive staining quantification from castrated LNCaP/AR for individual treatments groups: Vehicle control, SMAP-2 100mg/kg and SMAP-2 30mg/kg.

Supplementary Data from CIP2A Interacts with TopBP1 and Drives Basal-Like Breast Cancer Tumorigenesis

Supplementary Data from Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma

In cancer, kinases are often activated and phosphatases suppressed, leading to aberrant activation of signaling pathways driving cellular proliferation, survival, and therapeutic resistance. Although pancreatic ductal adenocarcinoma (PDA) has historically been refractory to kinase inhibition, therapeutic activation of phosphatases is emerging as a promising strategy to restore balance to these hyperactive signaling cascades. In this study, we hypothesized that phosphatase activation combined with kinase inhibition could deplete oncogenic survival signals to reduce tumor growth. We screened PDA cell lines for kinase inhibitors that could synergize with activation of protein phosphatase 2A (PP2A), a tumor suppressor phosphatase, and determined that activation of PP2A and inhibition of mTOR synergistically increase apoptosis and reduce oncogenic phenotypes in vitro and in vivo. This combination treatment resulted in suppression of AKT/mTOR signaling coupled with reduced expression of c-MYC, an oncoprotein implicated in tumor progression and therapeutic resistance. Forced expression of c-MYC or loss of PP2A B56α, the specific PP2A subunit shown to negatively regulate c-MYC, increased resistance to mTOR inhibition. Conversely, decreased c-MYC expression increased the sensitivity of PDA cells to mTOR inhibition. Together, these studies demonstrate that combined targeting of PP2A and mTOR suppresses proliferative signaling and induces cell death and implicates this combination as a promising therapeutic strategy for patients with PDA.Significance:These findings present a combinatorial strategy targeting serine/threonine protein phosphatase PP2A and mTOR in PDA, a cancer for which there are currently no targeted therapeutic options.

Supplementary Figure 7 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure 1 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure 2 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure 6 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure 4 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure Legends 1-7 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model