Blocking the cell cycle is a promising avenue for cancer therapy, with Cyclin-Dependent Kinase 2 (CDK2) emerging as a key target. However, in multiple cell types, CDK4/6 activity compensates for CDK2 inhibition and sustains the proliferative program, enabling CDK2 reactivation. Thus, we hypothesized that sensitivity to CDK2 inhibition is linked to the absence of this CDK4/6-mediated compensatory mechanism. Here we show that Cyclin E1-driven ovarian cancers often co-express the tumor suppressor p16, which inhibits CDK4/6. We show that ovarian cancer cells expressing p16 exhibit heightened sensitivity to CDK2 inhibitors and that depletion of p16 renders them significantly more resistant. Multiplexed immunofluorescence of 225 ovarian patient tumors reveals that at least 18% of tumors express high Cyclin E1 and high p16, a group that we expect to be particularly sensitive to CDK2 inhibition. Thus, p16 may be a useful biomarker for identifying the patients most likely to benefit from CDK2 inhibitors. Chance Sine, Lotte Watts, Brianna Fernandez, Jianxin Wang, Erik S. Knudsen, Agnieszka K. Witkiewicz, Sabrina L. Spencer. p16 confers sensitivity to CDK2 inhibitors in CCNE1-amplified ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5328.

Abstract Background Esophageal cancer (ESC) is an aggressive disease which often presents at an advanced stage. Despite trimodal therapy, 40–50% patients can develop metastatic disease by 18 months. Identification of patients at risk for metastatic spread is challenging with need for improved prognostication. We investigated whether the immune landscape of pretreatment tissue was associated with relapse in ESC patients. Methods Between April 2010 and October 2018, we identified 25 patients who had undergone trimodal therapy for ESC and had pretreatment biopsies suitable for analyses. We performed high-throughput multispectral immunofluorescence (mIF) analysis on formalin-fixed paraffin embedded biopsy samples. Analysis of 27 unique populations via immune and exhaustion mIF panels was performed and expression was normalized to total cell counts. Results Of the 25 patients analyzed, the median follow-up time was 23.9 months, during which 12 (48%) patients suffered a relapse with a median time to progression of 13.1 months. mIF revealed higher expression of HLA-DR (p = 0.019), CD8/LAG3 (p = 0.046), and CD8/CTLA4 (p = 0.027) among patients without relapse. Time to progression (TTP) and disease-specific survival (DSS) were stratified by median expression of each significant subpopulation and formally tested by the log-rank test. Higher than median expression of HLA-DR (p = 0.027), CD8/LAG3 (p = 0.039), and CD8/CTLA4 (p = 0.039) were significantly associated with TTP. Similarly, HLA-DR (p = 0.0069) and CD8/CTLA4 (p = 0.036) were significantly associated with improved DSS, whereas no significant observations were found with CD8/LAG3 (p = 0.11) expression. Stromal, but not tumoral expression of CD163 and CD163/PDL1 were significantly associated with improved TTP and DSS. Conclusions High expression of HLA-DR, CD8/CTLA4, and stromal expression of CD163 and CD163/PDL1 within pretreatment biopsy ESC samples was associated with significantly reduced rates of relapse. Increased presence of these markers suggests that an improved immune landscape is associated with less aggressive disease and may provide an opportunity for risk-based treatment strategies.

PURPOSE:The PENELOPE-B trial (ClinicalTrials.gov identifier: NCT01864746) recruited patients with hormone receptor+/human epidermal growth factor receptor 2- early breast cancer without a pathological complete response after taxane-containing neoadjuvant chemotherapy and at a high risk of relapse. Patients were randomly assigned (1:1) to receive 13 cycles of palbociclib once daily or placebo on days 1-21 in a 28-day cycle in addition to endocrine therapy (ET). PENELOPE-B did not show improved invasive disease-free survival (iDFS) after adding palbociclib to ET. This retrospective analysis investigated the impact of germline pathogenic variant (PV) status of BRCA1/2 and non-BRCA1/2 cancer predisposition genes on the outcomes of PENELOPE-B trial patients. METHODS:In total, 445 patients were sampled following a case-cohort design and 442 were analyzed for germline PVs. Statistical analyses were performed for time-to-event end points (iDFS, distant disease-free survival [DDFS], and overall survival [OS]). RESULTS:Of the 442 patients, 42 carried PVs in any cancer predisposition gene; 15 carried BRCA1/2 PVs. Irrespective of the treatment arms, PV status was not a prognostic factor. Regarding the treatment arms in BRCA1/2 PV carriers, numerically better 3-year outcomes were observed in the palbociclib arm (iDFS, 95%; DDFS, 95%; OS, 100%) than in the placebo arm (iDFS, 72.8%; DDFS, 72.8%; OS, 87.5%; hazard ratios palbociclib v placebo 0.349 [iDFS] and 0.562 [DDFS], not calculated for OS, too few events). In patients without BRCA1/2 PVs, the differences in 3-year outcomes were negligible. PVs in non-BRCA1/2 cancer predisposition genes did not influence the efficacy of palbociclib, although gene-specific effects could not be excluded. CONCLUSION:Patients with BRCA1/2 PVs had numerically better outcomes after palbociclib. However, the number of BRCA1/2 carriers was small. Larger randomized clinical trials should consider the PV status to further evaluate whether BRCA1/2 PV carriers benefit from cyclin-dependent kinase 4 and 6 inhibitor treatment.

Background: Nearly 30% of HR+ early breast cancer (eBC) patients (pts) treated with neoadjuvant chemotherapy (NACT) and surgery will experience BC recurrence, many with incurable distant metastatic disease. There is currently no blood-based biomarker that can identify pts with residual disease and at high risk of recurrence before and during adjuvant therapy. Thymidine kinase (TK) is an enzyme that plays a key role in DNA replication during cell division. The expression of TK is strongly linked to the cell cycle and measurement of TK activity (TKa) is a validated biomarker for both disease prognosis and therapy efficacy in metastatic breast cancer (mBC). We investigated the prognostic utility of TKa in eBC using patient serum samples from the PENELOPE-B study (NCT01864746). Methods: The PENELOPE-B phase III trial explored the addition of one year of palbociclib (P) to endocrine therapy (ET), in HR+, HER2- eBC pts at high risk of relapse after NACT. Serum samples were collected at baseline (BL), 6 months after starting therapy (C7), and end of treatment (EOT ∼ 13 months after starting therapy). Samples were analyzed retrospectively for TKa with the FDA cleared DiviTum® TKa assay (Biovica) using DuA (DiviTum unit of Activity) as the measuring unit. The cutoff for defining high vs low TKa was 250 DuA. In previous mBC studies, a TKa threshold value above 250 DuA was significantly associated with likelihood of disease progression. This same threshold value was applied to the PENELOPE-B sample set. TKa association with early invasive disease-free survival (iDFS) and distant disease-free survival (dDFS) was analyzed using restricted mean survival time (RMST) at a pre-specified timepoint of 1 year. The same method was also used to analyze the association of the clinical-pathologic stage- estrogen/grade (CPS-EG) score with early iDFS and dDFS using a cut-off score of <3 or >3. Results: 1251 pts enrolled in PENELOPE-B. BL TKa levels were analyzed in a sample set of 871 pts, 444 P+ET, 427 ET alone. Median follow-up time was 84 months. Among these 871 pts, 311 pts had an iDFS event (P + ET, 156; ET alone, 155) with 45 of these pts recurring in the first year. BL TKa ranged between 22 and 1025 DuA, with a mean value of 102 DuA, and a median of 81 DuA. BL TKa levels >250 DuA were significantly associated with both invasive and distant disease recurrence within the first 12 months of adjuvant therapy (RMST estimates for BL TKa>250 DuA vs BL TKa≤250 DuA: iDFS=9.6 vs 11.6 months, p=0.01, dDFS=9.9 vs 11.7 months, p= 0.02). In early relapse pts who had a serum sample taken at the time of study discontinuation, 18/22 (82%) had an increase in TKa at the time of relapse as compared to BL. The CPS-EG score when dichotomized as <3 or =4-5 was not prognostic for early relapse within a year (RMST estimates for CPS-EG >3 vs CPS-EG≤3, iDFS=11.3 vs 11.6 months, p=0.12, dDFS=11.3 vs 11.7 months, p= 0.08). TKa dynamic changes were also analyzed in 360 pts who had both C7 and EOT samples available for testing. Updated analysis looking at increases/decreases in TKa levels during therapy and association with recurrence, as well as treatment interaction will be presented. Conclusions: In a subset of pts from PENELOPE-B, TKa was detectable in all pts at BL and levels were significantly correlated with early disease recurrence where BL TKa values >250 DuA were prognostic for early relapse within the 1st year of adjuvant therapy. CPS-EG scores in contrast were not prognostic. Pts with early disease relapse also had an increase in TKa levels at the time of recurrence. These findings warrant further investigation of serum TKa as a non-invasive dynamic biomarker that could be used to assess in “real-time” the presence of actively proliferating disease in adjuvant BC pts and to monitor response to treatment via serial serum TKa testing. Citation Format: Amy J. Williams, Harry D. Bear, Carsten Denkert, Frederik Marmé, Erik Knudsen, Masey M. Ross, Seock-Ah Im, Angela DeMichele, José Angel García Sáenz, Agnieszka Witkiewicz, Laura Van’t Veer, Sung-Bae Kim, Zhe Zhang, Nicholas Turner, Federico Rojo, Martin Filipits, Lesley-Ann Martin, Olga Valota, Peter A. Fasching, Christian Schem, Nicole Mc Carthy, Toralf Reimer, Bärbel Felder, Karsten Weber, Valentina Nekljudova, Sibylle Loibl. Evaluation of proliferation biomarker serum thymidine kinase activity and prediction of early relapse in HR positive HER2 negative high risk early breast cancer: Analysis from the PENELOPE-B trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-09-06.

566 Background: The concept of intrinsic subtyping has been an important step for understanding of breast cancer (BC) as a heterogenous disease. However, these subtypes are not adapted to therapy-induced molecular plasticity. We have evaluated a high-risk luminal BC clinical trial cohort to identify new additional adaptive BC subtypes based on molecular alterations induced during neoadjuvant chemotherapy (NACT). Methods: A total of 1250 ER+/HER2- BC patients (pts) with residual disease after NACT and increased risk (CPS-EG score of ≥3 or 2 with ypN+) were randomized into the PENELOPE-B (NCT01864746) trial to receive palbociclib or placebo. Biomarker analysis was performed in 1411 pre- or postNACT tumor samples including 540 paired samples using the HTG EdgeSeq Oncology Biomarker Panel targeting 2549 genes (HTG Molecular Diagnostics Inc.), with assessment of the absolute assignment of breast cancer intrinsic molecular subtype (AIMS). For a subcohort of 29 pts, we evaluated triplicate samples before and after NACT as well as at the time of metastatic disease. Results: In paired biopsies, a total of 335 genes were significantly different between the pre- and the postNACT cohort. With hierarchical unsupervised clustering of these 335 genes, we characterized five different tumor subtypes with highly significant iDFS survival differences (p<0.0001, n=539). We identified two large groups of tumors with excellent prognosis (adaptive cluster AC1 and AC2, together n=284, 6 events), as well as two groups with a poor prognosis; AC-3 (n=163, 82 events) and AC-4 (n=78, 29 events). The worst prognosis was observed in a small group of tumors with a pre-NACT Basal/HER2E AIMS subtype (n=14, 11 events). AC1 and AC3 are enriched for tumors with an AIMS subtype change from LumB to LumA during NACT, with improved iDFS for AC1. AC2 and AC4 are enriched for tumors with a constant LumA AIMS subtype before and after NACT, with improved iDFS for AC2. The low-risk adaptive subtypes AC1 and AC2 cover a total of 52.7% of pts with a combined event rate of 2.1%. In contrast, the best pretherapeutic conventional AIMS-subtype, preNACT LumA, covers 51.7% of pts, but still has an event rate of 15.4%. This suggests that adaptive subtypes are superior to classical AIMS subtypes for prediction of survival after NACT in luminal BC. Conclusions: Classical approach of intrinsic subtyping relies on constant gene expression, limiting its scope. Adaptive subtyping can complement the classical approach, focusing on those genes that are changed during therapy. Comparison of paired samples before and after therapy is superior to classical subtyping of baseline samples. This approach identified large subgroups of pts with excellent prognosis after NACT despite clinical high risk. This can be an important step to focus on high-risk pts for new neoadjuvant and post-neoadjuvant therapy concepts.

Anti-EGFR therapy has the potential to increase antitumor immune responses. Therapeutic strategies targeting EGFR and immune checkpoints may benefit patients (pts) with mCRC. We conducted a phase Ib/II clinical trial of cetuximab with pembrolizumab in RAS wild-type (RASwt) mCRC; the primary efficacy results of the trial have been reported (Fountzilas et al ESMO GI 2020). Herein, we present correlative tissue analysis. Tumor biopsies were obtained baseline (pre-Tx) and after 12 weeks (post-Tx); multiplex immunohistochemistry for PD-L1, T-cell exhaustion (TIM3, CTLA4, LAG3) and activation (OX40) markers was performed. T-cells were characterized as partially activated-1 (OX40+/PD-L1-, 1/3 exhaustion markers+); partially activated-2 (OX40+/PD-L1-, 2/3 exhaustion markers+); partially activated-3 (OX40+/PD-L1-, 3/3 exhaustion markers+); exhausted-1 (OX40-/PD-L1-, 1/3 exhaustion markers+); exhausted-2 (OX40-/PD-L1-, 2/3 exhaustion markers+); and exhausted-3 (OX40-/PD-L1-, 3/3 exhaustion markers+). Forty-two pts were enrolled; 14 with matched pre-Tx and post-Tx biopsies, 24 with pre-Tx only. PD-L1 expression was higher in pre-Tx metastasis (M) vs. primary tumor (PT) in both tumor and stromal cells within the microenvironment (TME). The no. of activated T-cells was higher pre-Tx in PT compared to M. The no. of exhausted-2 T-cells in pre-Tx was higher in M vs. PT. The no. of activated T-cells and partially activated-1 T-cells was higher in post-Tx vs. pre-Tx M. There was no change in the no. of exhausted T-cells on post-Tx. PD-L1 decreased on tumor cells and increased in the TME on post-Tx. Temporospatial heterogeneity in the immune landscape of the TME was evident in RASwt mCRC. With the limitation of small sample size, we observed increased PD-L1 expression and exhausted T-cells and decreased activated T-cells in M compared to PT. There was an increase in activated T-cells post-Tx, but the no. of exhausted T-cells was similar, potentially explaining the lack of additive benefit from pembrolizumab. Evaluating the PT in mCRC might not be reflective of immunosuppression in M. Combinations with mAb targeting other T-cell exhaustion pathways may be necessary to improve the antitumor efficacy of the EGFR-based regimens.

Abstract This abstract was withdrawn by the authors.

Abstract 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 IC50 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.

Purpose: HER2+ breast cancer is treated using agents that target the amplified receptor. Such drugs are effective; however, recurrence and progression can occur in spite of multiple HER2-targeted therapies. This study evaluated the safety and potential efficacy of the combination of TDM-1 and palbociclib in previously treated metastatic HER2+ breast cancer. Background: Preclinical data indicate that the CDK4/6 inhibition suppresses proliferation of HER2+ tumor models, including those resistant to HER2-targeted therapies. The action of CDK4/6 inhibitors is distinct from HER2-targeted agents. In particuar T-DM1 elicits a cytotoxic response; while CDK4/6 inhibition prevents outgrowth of tumors that survive T-DM1. These data were used to develop scheduling for a study to evaluate the safety and preliminary efficacy of palbociclib combined with TDM-1 in advanced HER2+ breast cancer. Experimental design: A standard 3X3 design Phase I trial design was applied. Centrally confirmed HER2+ and RB-intact advanced breast cancer patients, including those previously exposed to TDM-1, were enrolled. Patients received TDM-1 (3.6 mg/kg) IV every 21 days and palbociclib orally on days 5-18 of each cycle. Dose escalation occurred in 100, 150 and 200 mg cohorts. Of 18 patients screened, two were excluded due to lack of HER2-positivity or observed RB-deficiency. Nine patients were in the dose escalation and 44% (4/9) had received prior TDM-1 therapy. Results: Predominant study drug related toxicities were hematologic, with 33% of patients experiencing grade 3 thrombocytopenia. Grade 3 neutropenia related to palbociclib was seen in 33% of patients at 100 mg, 66% at 150 mg, and 100% at 200 mg dose level. Toxicity resolved with dose interruption. Best observed responses included 3 partial responses, 5 stable diseases, and 1 progressive disease. Median cycles was 8, with average time on study 193 days. Of four patients previously relapsing on TDM-1, two had stable disease and one partial response. Conclusions: The TDM-1 and palbocicilb combination was well tolerated with reversible hematologic toxicity and evidence of clinical efficacy. The recommended dose for further study is TDM-1 (3.6 mg/kg) day 1, with 150 mg of palbociclib on days 5-18 of a 21 day cycle. Clinical trial identification: NCT01976169 Legal entity responsible for the study: UT Southwestern Medical Center Funding: NIH and Pfizer Disclosure: E.S. Knudsen: Received research funding, support for clinical studies, and served on advisory panels for Eli Lilly, Pfizer, and Novartis which have CDK4/6 inhibitors in clinical development. The current clinical trial was supported by Pfizer. All other authors have declared no conflicts of interest.

Purpose: Triple negative breast cancer (TNBC) is a heterogeneous disease for which more targeted interventions are needed. Here we employed an integrated combination of biomarker analyses and targeted drug screening to define actionable subtypes of TNBC. Methods: A cohort of 218 TNBC cases annotated for survival were analyzed for the expression of multiple markers by immunohistochemistry (IHC). Markers included were associated with immune function (e.g. CD163), cell cycle (e.g. RB), and defined subtypes (e.g. vimentin and AR). Affinity propogation clustering and random forest approaches defined specific subtypes of TNBC that were highly enriched for prognosis. Models that mimicked these subtypes were evaluated for sensitivity to established cancer drugs using high-throughput approaches and in selected combinations based on marker profile. Results: Using unsupervised methods, IHC defined classes of TNBC with particularly preferable and poor prognosis that remained significant in multivariate analysis. For example, tumors with combined loss of RB and PTEN had a 5-year survival of >85% (p < 0.01), while mesenchymal tumors with expression of vimentin exhibited a 5-year survival of <25% (p < 0.005). Two exclusive classes of TNBC (RB-negative and AR-positive) that represent ∼50% of cases were evaluated for therapeutic sensitivities. RNA sequencing of clinical cases demonstrated that tumors deficient in RB express high levels of genes activating CDK5, PLK1, AURK, CHK kinases. Concordantly, RB-deficient tumor models are selectively sensitive to agents targeting these kinases. In contrast, AR positive tumors retain RB and exhibit low proliferation index in clinical cases. TNBC models of AR positive disease were particularly sensitive to CDK4/6 inhibitors alone and in combination with AR-antagonists. Conclusions: These data indicate that with a limited collection of markers that it is possible to delineate prognostic subtypes of TNBC. Within TNBC cases, select classes of tumors have distinct vulnerabilities that can be targeted therapeutically. Disclosure: All authors have declared no conflicts of interest.

Purpose: In general cells that exited the cell cycle are resistant to conventional cytotoxic therapies. This represents one of the seminal challenges in eliminating the reservoir for disease recurrence that is particularly problematic in the context of ER+ breast cancer. Here we investigated metabolic features and resultant therapeutic sensitivities that are unique to induced dormancy. Methods: Therapy induced dormancy was mediated using ER antagonists (e.g. Fulvestrant) and CDK4/6 inhibitors (e.g. PD-0332991) in models of ER+ positive breast cancer. These models were subjected to Mass-spectromety and metabolomics analysis to define the metabolic state in dormancy. Drug screens were used to define unique sensitivities that are imparted in non-proliferative cancer cells to delineate novel means of eliminate dormant cancer cells. Results: In models of ER+ breast cancer endocrine therapy and CDK4/6 inhibition were effective at suppressing proliferation, but elicited surprisingly distinct effects on tumor metabolism. Endocrine therapy limited glycolytic and oxidative metabolism, while CDK4/6 inhibition resulted in increased metabolic function. Treatment with CDK4/6 inhibitors resulted in significantly increased mitochondrial mass and oxidative metabolism. Combination studies indicated that fulvestrant has a dominant effect on metabolism over CDK4/6 inhibitors, suggesting a novel basis for cooperation. Drug screening showed that both fulvestrant and CDK4/6 inhibitors limited the effectiveness of a host of conventional cytotoxic agents, yet are additive with multiple clinically employed targeted agents. Due to the enhanced metabolic flux in CDK4/6 arrested models, unique sensitivities to agents targeting metabolic functions were uncovered. Conclusions: Dormant cells that have exited the cell cycle exhibit distinct metabolic features. These unique aspects of tumor biology can be targeted selectively using novel combinations in concert with endocrine therapy and/or CDK4/6 inhibitors. Disclosure: All authors have declared no conflicts of interest.

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.

Purpose: In spite of the efficacy of Her2-targeted therapies, recurrence and progression remain a challenge for treatment of advanced Her2 positive breast cancer. CDK4/6 controls a key pathway downstream of Her2, hence inhibition of these kinases represents a therapeutic approach to augment the effectiveness of standard therapies. Methods: A combination of cell culture, mouse models, and human primary tumor explants were used to evaluate the therapeutic effect of the CDK4/6 inhibitor PD-0332991 as a single agent and to define markers of response. Parallel studies evaluated the mechanisms of action in combination with the Her2-targeted agents Lapatinib and T-DM1. Results: CDK4/6 inhibition resulted in profound cytostatic arrest, induction of senescence, and inhibition of invasive properties in Her2-positive cell culture models. These data were recapitulated with significant suppression of Ki67 in the MMTV-Her2 mouse model (p < 0.05) and Her2-positive xenografts (p < 0.01). Furthermore, in a series of more than 20 primary breast tumor explants, treatment with PD-0332991 resulted in a greater than 5-fold suppression of the Ki67 (p < 0.01). These effects of PD-0332991 were dependent on an intact RB-pathway, and consonantly, loss of RB and high-levels of p16 were associated with resistance to CDK4/6 inhibition. In models of acquired resistance to Her2-targeted therapies Cyclin D1 was inappropriately activated, and PD-0332991 treatment was effective at blocking proliferation by targeting this common pathway driving resistance. Combination studies carried out in cell lines and primary tumor explants illustrated that PD-0332991 provides a complementary mechanism of action to T-DM1, and efficiently suppresses the proliferation of residual Her2-positive tumor cell populations that survive T-DM1. Conclusions: CDK4/6 is a viable therapeutic target in HER2-positive breast cancer models that functions downstream of Her2. Tissue based markers are available to direct rational utilization of PD-0332991. Clinical studies of CDK4/6 inhibition in combination with Her2-targeted therapies have commenced. Disclosure: E. Knudsen: The author is on an advisory board and receives sponsored research funding from Pfizer. All other authors have declared no conflicts of interest.