Valerie M. Jansen

Treatment of Triple-Negative Breast Cancer with TORC1/2 Inhibitors Sustains a Drug-Resistant and Notch-Dependent Cancer Stem Cell Population.

Approximately 30% of triple-negative breast cancers (TNBC) harbor molecular alterations in PI3K/mTOR signaling, but therapeutic inhibition of this pathway has not been effective. We hypothesized that intrinsic resistance to TORC1/2 inhibition is driven by cancer stem cell (CSC)-like populations that could be targeted to enhance the antitumor action of these drugs. Therefore, we investigated the molecular mechanisms by which PI3K/mTOR inhibitors affect the stem-like properties of TNBC cells. Treatment of established TNBC cell lines with a PI3K/mTOR inhibitor or a TORC1/2 inhibitor increased the expression of CSC markers and mammosphere formation. A CSC-specific PCR array revealed that inhibition of TORC1/2 increased FGF1 and Notch1 expression. Notch1 activity was also induced in TNBC cells treated with TORC1/2 inhibitors and associated with increased mitochondrial metabolism and FGFR1 signaling. Notably, genetic and pharmacologic blockade of Notch1 abrogated the increase in CSC markers, mammosphere formation, and in vivo tumor-initiating capacity induced by TORC1/2 inhibition. These results suggest that targeting the FGFR-mitochondrial metabolism-Notch1 axis prevents resistance to TORC1/2 inhibitors by eradicating drug-resistant CSCs in TNBC, and may thus represent an attractive therapeutic strategy to improve drug responsiveness and efficacy.

Kinome wide functional screen identifies role of Polo-like kinase 1 (PLK1) in hormone-independent, ER-positive breast cancer

Estrogen receptor (ER) α-positive breast cancers initially respond to antiestrogens but eventually become estrogen independent and recur. ER(+) breast cancer cells resistant to long-term estrogen deprivation (LTED) exhibit hormone-independent ER transcriptional activity and growth. A kinome-wide siRNA screen using a library targeting 720 kinases identified Polo-like kinase 1 (PLK1) as one of the top genes whose downregulation resulted in inhibition of estrogen-independent ER transcriptional activity and growth of LTED cells. High PLK1 mRNA and protein correlated with a high Ki-67 score in primary ER(+) breast cancers after treatment with the aromatase inhibitor letrozole. RNAi-mediated knockdown of PLK1 inhibited ER expression, estrogen-independent growth, and ER transcription in MCF7 and HCC1428 LTED cells. Pharmacologic inhibition of PLK1 with volasertib, a small-molecule ATP-competitive PLK1 inhibitor, decreased LTED cell growth, ER transcriptional activity, and ER expression. Volasertib in combination with the ER antagonist, fulvestrant, decreased MCF7 xenograft growth in ovariectomized mice more potently than each drug alone. JUNB, a component of the AP-1 complex, was expressed 16-fold higher in MCF7/LTED compared with parental MCF7 cells. Furthermore, JUNB and BCL2L1 (which encodes antiapoptotic BCL-xL) mRNA levels were markedly reduced upon volasertib treatment in MCF7/LTED cells, while they were increased in parental MCF7 cells. Finally, JUNB knockdown decreased ER expression and transcriptional activity in MCF7/LTED cells, suggesting that PLK1 drives ER expression and estrogen-independent growth via JUNB. These data support a critical role of PLK1 in acquired hormone-independent growth of ER(+) human breast cancer and is therefore a promising target in tumors that have escaped estrogen deprivation therapy.

Kinome-wide RNA interference screen reveals a role for PDK1 in acquired resistance to CDK4/6 inhibition in ER-positive breast cancer

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) small-molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, yield sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a key modifier of ribociclib sensitivity in estrogen receptor-positive MCF-7 breast cancer cells. Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase in the levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell-cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and to restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib, in combination with GSK2334470 or the PI3Kα inhibitor alpelisib, decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K-PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. Cancer Res; 77(9); 2488-99. ©2017 AACR.

Is There a Future for AKT Inhibitors in the Treatment of Cancer

An AKT inhibitor plus an antiestrogen exhibited no significant clinical activity in patients with ER+/HER2− breast cancer despite laboratory studies supporting an antitumor effect for both drugs combined. These results raise concerns about the development of AKT inhibitors in unselected patients whose tumors have unknown dependence on the PI3K/AKT pathway. Clin Cancer Res; 22(11); 2599–601. ©2016 AACR. See related article by Ma et al., p. 2650

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK4/6 inhibitor palbociclib

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor‐positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib‐induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib‐induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence‐like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse‐free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.

Abstract P3-03-05: PI3K/PDK1 mediates resistance to CDK4/6 inhibitors through dysregulation of S-phase cyclins/cyclin dependent kinases (CDKs)

Background: CDK4/6 inhibitors in combination with antiestrogens are approved for the treatment of ER+ advanced breast cancer. However, not all patients benefit from CDK4/6 inhibitors, underscoring the need to develop therapeutic strategies to circumvent de novo and acquired drug resistance. Methods: ER+ breast cancer cells (MCF-7, T47D, HCC1428, and HCC1500) were made resistant to increasing doses to the CDK4/6 inhibitor ribociclib (LEE011; Novartis). LEE011-resistant cells were characterized by 2D/3D growth, cell cycle, and immunoblot analyses. GSK2334470 (PDK1 inhibitor) and dinaciclib (CDK2 inhibitor) were used to modify resistance to ribociclib. PDK1 and pS6 immunohistochemistry (IHC) were performed on primary human tumor explants treated ex vivo with palbociclib. Results: Resistant cell lines (MCF-7/LR, T47D/LR, HCC1428/LR, and HCC1500/LR) exhibited an IC 50 at least 20-fold higher than that of their parental cells. They displayed cross-resistance to the CDK4/6 inhibitors palbociclib and abemaciclib. Immunoblot analysis of ribociclib-resistant cells showed increased levels of 3-phosphoinositide dependent protein kinase 1 (PDK1), S227 pRSK2 (target of PDK1), T308 pAKT (target of PDK1), and pS6 (downstream effector of the PDK1 target p70S6K), compared to parental drug sensitive cells. PDK1 is a master kinase that functions downstream of phosphoinositide 3-kinase (PI3K) and is crucial for the activation of AKT and many other AGC kinases including PKC, S6K, SGK, and RSK. Primary tumor explants treated ex vivo with palbociclib for 96 h also exhibited upregulation of PDK1 and pS6 by IHC. Cell cycle analysis revealed that CDK4/6 inhibition failed to induce G1 arrest, a reduction in S phase, and senescence in MCF-7/LR and T47D/LR compared to parental cells. Progression into S phase in the presence of ribociclib suggested upregulation of S-phase cyclins/CDKs. Indeed, the resistant cells exhibited significantly higher levels of pCDK2, cyclin A, cyclin E and S477/T479 pAKT, a CDK2-dependent phosphorylation of AKT required for full kinase activity and limited to the S-phase of the cell cycle. Pharmacological inhibition of PDK1 (with GSK2334470) or CDK2 (with dinaciclib) re-sensitized the ribociclib-resistant cells to CDK4/6 inhibitors. However, ribociclib/GSK2334470 inhibited MCF-7/LR and T47D/LR cell proliferation better than ribociclib/dinaciclib. Further, ribociclib/GSK2334470 but not ribociclib/dinaciclib completely abrogated pRb, pS6, pRSK2, pCDK2, cyclin A, and cyclin E, suggesting the PI3K/PDK1 pathway mediates acquired resistance to CDK4/6 inhibitors through dysregulation of the cell cycle. Consistent with these data, ribociclib/GSK2334470 inhibited growth of established MCF-7 xenografts in nude mice, significantly more potently than each drug alone. Conclusions: These data support a critical role for PI3K/PDK1 in acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells. Co-targeting of PI3K/PDK1 and CDK4/6 may overcome resistance to CDK4/6 inhibitors and is worthy of further translational and clinical investigation in patients with ER+ breast cancer. Citation Format: Jansen VM, Formisano L, Witkiewicz A, Estrada MV, Sanchez V, Dugger TC, Knudsen ES, Arteaga CL. PI3K/PDK1 mediates resistance to CDK4/6 inhibitors through dysregulation of S-phase cyclins/cyclin dependent kinases (CDKs) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-05.

Abstract PD2-06: Inhibition of 3-phosphoinositide dependent protein kinase 1 (PDK1) synergizes with CDK4/6 inhibitors against ER-positive breast cancer

Background: Dysregulation in cell cycle checkpoints is common in cancer. Small molecule inhibitors that target the CDK4/6/cyclinD1 pathway of the cell cycle are in clinical development. Recently the combination of the CDK4/6 inhibitor palbociclib and the aromatase inhibitor letrozole was approved for the treatment of post-menopausal women with ER+/HER2- advanced breast cancer. However, not all patients benefit from CDK4/6 inhibitors and a significant fraction of them eventually progress on these agents, underscoring the need to develop potent therapeutic strategies to circumvent drug resistance. Methods: We performed a high-throughput RNA interference (RNAi) kinome screen targeting 720 kinases to identify targetable molecules whose inhibition, in combination with the CDK4/6 inhibitor LEE011 (ribociclib), induced synthetic lethality in MCF7 ER+ breast cancer cells. PDK1 RNAi oligonucleotides and the PDK1 inhibitor GSK2334470 in combination with each of the CDK4/6 inhibitors, palbociclib and LEE011, were tested against ER+ breast cancer cells. In vivo anti-tumor efficacy of LEE011 and GSK2334470 was assessed in ovariectomized athymic nude mice bearing MCF7 xenografts. Results: A siRNA kinome screen identified PDK1 as the top RNA whose downregulation sensitized MCF7 cells to CDK4/6 inhibitors. This was confirmed with independent siRNAs in ER+ MCF7, T47D, HCC1428 and HCC1500 breast cancer cells. Pharmacological inhibition of PDK1 with the ATP-competitive, small molecule inhibitor GSK2334470 in combination with each of the CDK4/6 inhibitors, LEE011 and palbociclib, synergistically inhibited proliferation and increased apoptosis of MCF7 and T47D cells (combination index 0.19-0.89). LEE011-resistant MCF7 and T47D cells were generated by chronic treatment with doses of LEE011 up to 1 µM. Drug-resistant cells displayed increased levels of PDK1, phosphorylated Rb, and phosphorylated S6 ribosomal protein (pS6), an effector of the PDK1 substrate p70S6K, compared to parental drug-sensitive cells. Inhibition of PDK1 with siRNA or GSK2334470 re-sensitized the LEE011-resistant cells to the CDK4/6 inhibitors. Genetic (RNAi) and pharmacological inhibition of PDK1 (with GSK2334470) abrogated pS6 levels whereas inhibition of AKT with the small molecule inhibitor MK2206 did not affect pS6 levels, suggesting PDK1 can induce resistance to CDK4/6 inhibitors via p70S6K/pS6 signaling in an AKT-independent manner. The effects observed in cell lines in culture were recapitulated in vivo using MCF7 xenografts established in ovariectomized nude mice in the absence of estrogen supplementation. Treatment with GSK2334470 and LEE011 induced tumor regressions (8/8 tumors by RECIST criteria) more potently than either drug alone. Conclusions: These data support a critical role of PDK1 in mediating acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells. Co-targeting of the PDK1 and CDK4/6 pathways may overcome resistance to CDK4/6 inhibitors and is worthy of further translational and clinical investigation in patients with ER+ breast cancer. Citation Format: Jansen VM, Bhola NE, Bauer JA, Formisano L, Moore P, Koch J, Arteaga CL. Inhibition of 3-phosphoinositide dependent protein kinase 1 (PDK1) synergizes with CDK4/6 inhibitors against ER-positive breast cancer. [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 PD2-06.

Abstract 1945: TORC inhibitors increase the cancer stem cell (CSC) population and Notch signaling in triple negative breast cancer

Tumor-initiating cells (TICs) or cancer stem cells (CSCs) are resistant to chemotherapy and have been associated with metastatic recurrences and poor patient outcome particularly among patients with triple negative breast cancer (TNBC). Genomic and proteomic data have indicated more than 30% of TNBC patients have PI3K/mTOR pathway lesions making this pathway a promising therapeutic target. Recent publications have demonstrated mechanisms of resistance (JAK2/STAT5 and MYC amplification) to PI3K pathway inhibitors. We hypothesized that resistance to TORC inhibition is due to the survival of a CSC population and that targeting pathways that sustain these cells can provide a significant therapeutic benefit. Treatment of TNBC cell lines with the PI3K/mTOR inhibitor NVP-BEZ235 or the TORC1/2 inhibitor MLN128 resulted in a significant reduction of proliferation in vitro. However, we observed that both BEZ235 and MLN128 enriched for a CSC population as assessed by FACS analysis of cancer stem-like markers and mammosphere formation. This observation was specific to TNBC cell lines since BEZ235 and MLN128 significantly abrogated the CSC population in ER+ (MCF7) and HER2+ (HCC1954) breast cancer cell lines. To determine the mechanisms involved in this CSC enrichment we used a Stem Cell specific PCR Array. We observed an increase in Notch1, FGF1 and ABCG2 mRNA levels in TNBC cells treated with BEZ235 and MLN128. Treatment with these inhibitors also increased the expression of the active Notch intracellular domain, the Notch ligand Jagged1, and the Notch1 target genes Hes1 and Hey1 by qRT-PCR and transcriptional reporter activity. In addition to Hes1 and Hey1, c-myc, another Notch target gene, expression was augmented in 2 of the 3 TNBC cell lines tested. Treament with the γ-secretase inhibitor, DAPT and transfection with Notch1 siRNA oligonucleotides abrogated BEZ235 and INK128-mediated enrichment of CSC populations as measured by FACS analysis and mammosphere formation assays. To determine whether inhibition of either TORC1 or TORC2 enriched for the CSC population, we used RNAi against Rictor (TORC2), Raptor (TORC2) or both. We observed that only the combined knockdown of Rictor and raptor increased the CSC population in TNBC cell lines. These observations suggest that treatment of TNBC harboring PI3K pathway aberrations with TORC1/2 inhibitors results in an initial reduction of tumor burden but do not eradicate the drug-resistant, slow cycling CSC population driven by Notch signaling. Thus, combination of a Notch inhibitor with TORC1/2 inhibitors and chemotherapy may be an effective therapeutic strategy to decrease primary tumor growth and prevent recurrences in patients with TNBC. Citation Format: Neil E. Bhola, Valerie Jansen, Carlos Arteaga. TORC inhibitors increase the cancer stem cell (CSC) population and Notch signaling in triple negative 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 1945. doi:10.1158/1538-7445.AM2014-1945

Abstract S3-03: Nuclear FGFR1 interaction with estrogen receptor (ER) α is associated with resistance to endocrine therapy in ER+/FGFR1-amplified breast cancer

Background: Estrogen receptor (ER)-positive breast cancers (BC) initially respond to antiestrogens but eventually become hormone-independent and recur. FGFR1 is amplified in ∼10% of ER+ BC and is associated with early recurrence on antiestrogen therapy. Notably, one third of FGFR1-amplified tumors have simultaneous amplification of CCND1, FGF3, FGF4 and FGF19 on chromosome 11q12-14. Herein, we investigated the mechanisms by which FGFR1 amplification confers resistance to antiestrogen therapy in ER+ BC cells. Results: We performed whole exome sequencing in tumor biopsies from 130 patients with an operable ER+/HER2- BC who had received letrozole for 10-21 days prior to surgery. Tumors were categorized by the natural log (ln) of post-letrozole Ki67 as sensitive (ln ≤1 or ≤2.7% Ki67+ cells; n=68) or resistant (ln ≥2 or ≥7.4%; n=18). We found amplifications in FGFR1 and/or 11q12-14 in 6/11 (55%) resistant tumors compared with 5/34 (15%) in sensitive tumors (p=0.006); all cases were confirmed by FGFR1-fluorescence in situ hydridization (FISH). Resistant tumors with FGFR1 and/or 11q12-14-amplification showed a marked increase in nuclear FGFR1 with letrozole. ER+/FGFR1-amplified CAMA1 and MDA134 cell lines also exhibited co-localization of ER and FGFR1 in the nucleus. Cell proliferation was partially reduced by estrogen deprivation, and FGFR1 siRNA further reduced cell growth in hormone-depleted medium. We generated CAMA1 and MDA134 cells resistant to long-term estrogen deprivation (LTED). These cells exhibited overexpression of FGF3/4/19 and ERα with a concomitant increase in ligand-independent ER transcriptional activity and growth. An ER-FGFR1 interaction was observed in the nucleus and cytosol of CAMA1 parental cells with enhanced interaction in CAMA1 LTED cells. Genetic (with siRNA) and pharmacologic (with lucitinib) inhibition of FGFR1 reduced a) nuclear localization of FGFR1; b) ER transcriptional activity; and c) cell proliferation. Nuclear localization and ER-FGFR1 interaction were disrupted by a kinase-deficient FGFR1. Conversely, addition of FGF3 ligand stimulated ER-FGFR1 interaction and ER transcriptional activity, suggesting FGFR activation can regulate ER function. Inhibition of FGF receptor-specific substrate (FRS2), a principal mediator of FGFR1 signal transduction to the MAPK and PI3K pathways, with siRNA or pharmacologic inhibition of PI3K with buparlisib or MEK with GSK1120212 did not reduce ER transcriptional activity suggesting that, in ER+/FGFR1-amplified cancer cells, ER function is not modulated by FGFR signal transducers. Finally, using chromatin immunoprecipitation (ChIP) we showed that FGFR1 binds directly to estrogen response elements (ERE). This association was reduced with lucitanib. We are currently investigating genes modulated by ER/FGFR1 in ER+ BC and the in vivo anti-tumor efficacy of dual inhibition of FGFR1 and ER in ER+/FGFR1-amplified patient-derived breast cancer xenografts. Conclusions: These data support a critical role of ER and FGFR1 interaction in endocrine resistance in ER+/FGFR1-amplified breast cancer. Targeting of FGFR1 in combination with antiestrogens may abrogate resistance to endocrine therapy in these tumors and is worthy of clinical investigation. Citation Format: Formisano L, Young CD, Bhola NE, Bulen B, Estrada VM, Wagle N, Van Allen E, Red Brewer ML, Jansen VM, Guerrero AL, Giltnane JM, Strcker T, Arteaga CL. Nuclear FGFR1 interaction with estrogen receptor (ER) α is associated with resistance to endocrine therapy in ER+/FGFR1-amplified breast cancer. [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 S3-03.

Abstract 2844: RNA interference kinome-wide screen reveals a role for PDK1 in acquired resistance to CDK4/6 inhibition ER-positive breast cancer

Background: Small molecule inhibitors that target the CDK4/6/cyclinD1 pathway are in clinical development. Clinical trials combining the CDK4/6 inhibitor pallbociclib and the aromatase inhibitor letrozole have demonstrated significantly improved clinical outcomes in patients with ER-positive breast cancer. This combination is likely to be approved for the treatment of patients with this breast cancer subtype. However, as for other targeted therapies, development of resistance to CDK4/6 inhibitors in a significant fraction of patients is anticipated. Therefore, there is a need to develop potent therapeutic strategies to circumvent drug resistance. Methods: We performed a high-throughput RNAi kinome screen targeting 720 kinases to identify potentially targetable molecules whose inhibition in combination with the CDK4/6 inhibitor LEE011 induced synthetic lethality in MCF7 ER+ breast cancer cells. Results: The sensitivity index (SI) score, which measures the influence of siRNA-induced gene knockdown on drug sensitivity, was calculated for each siRNA in MCF7 cells treated with LEE011 (IC 50 , 144 nM). A cutoff of SI >0.15 (2 standard deviations above the mean) was used for hit selection. Individual knockdown of 15 (2.1%) kinases sensitized MCF7 cells to the CDK4/6 inhibitor (p Conclusions: These studies suggest that PDK1 inhibition can overcome resistance to the CDK4/6 inhibitor LEE011 and offer a rationale for further translational and clinical investigation of combinations of CDK4/6 and PDK1 inhibitors in ER+ breast cancer. Citation Format: Valerie M. Jansen, Neil E. Bhola, Joshua A. Bauer, Carlos L. Arteaga. RNA interference kinome-wide screen reveals a role for PDK1 in acquired resistance to CDK4/6 inhibition ER-positive breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2844. doi:10.1158/1538-7445.AM2015-2844