Palbociclib plus endocrine therapy significantly enhances overall survival of HR+/HER2- metastatic breast cancer patients compared to endocrine therapy alone in the second-line setting: A large institutional study.

Cyclin-dependent-kinase-4/6 inhibitor (CDK4/6i) plus endocrine therapy (ET) is standard of care for patients with advanced hormone receptor (HR)-positive, HER2-negative breast cancer (BC). The Breast Medical Oncology database at MD Anderson Cancer Center (MDACC) was analyzed to assess effectiveness of the CDK4/6i palbociclib plus ET compared to ET alone. From a total of 5402 advanced HR+ HER2- BC patients referred to MDACC between 1997 and 2020, we identified eligible patients who received palbociclib in combination with first-line (n = 778) and second-line (n = 410) ET. We further identified "control" patients who received ET alone in the first-line (n = 2452) and second-line (n = 1183) settings. Propensity score matching analysis was conducted to balance baseline demographic and clinical characteristics between palbociclib and control cohorts to assess the effect of palbociclib treatment on progression-free survival (PFS) and overall survival (OS). For propensity-matched-cohort in the first-line setting (n = 708), palbociclib group had significantly longer median PFS (17.4 vs 11.1 months; P < .0001) compared to controls. Median OS (44.3 vs 40.2 months) did not show a statistically significant benefit in the first line setting. However, in the second-line setting, with 380 propensity-matched-cohort, the palbociclib group had significantly longer PFS (10 vs 5 months, P < .0001) as well as OS (33 vs 24 months; P < .022), compared to controls. We conclude that in this single center analysis of a large cohort of metastatic HR+ HER2- BC patients, palbociclib in combination with ET was associated with improved PFS in both first-line and second-line settings and OS in the second-line setting compared to ET alone cohort.

Cytoplasmic Cyclin E Expression Predicts for Response to Neoadjuvant Chemotherapy in Breast Cancer.

BACKGROUND: Pathologic complete response (pCR) has been shown to be associated with favorable outcomes in breast cancer. Predictors of pCR could be useful in guiding treatment decisions regarding neoadjuvant therapy. The objective of this study was to evaluate cyclin E as a predictor of response to neoadjuvant chemotherapy in breast cancer. METHODS: Patients (n = 285) with stage II-III breast cancer were enrolled in a prospective study and received neoadjuvant chemotherapy with anthracyclines, taxanes, or combination of the two. Pretreatment biopsies from 190 patients and surgical specimens following chemotherapy from 192 patients were available for immunohistochemical analysis. Clinical and pathologic responses were recorded and associated with presence of tumor infiltrating lymphocytes, cyclin E, adipophilin, programmed cell death-ligand 1, and elastase staining and other patient, tumor and treatment characteristics. RESULTS: The pCR rate was significantly lower in patients with cytoplasmic cyclin E staining compared with those who had no cyclin E expression (16.1% vs 38.9%, P = 0.0005). In multivariable logistic regression analysis, the odds of pCR for patients who had cytoplasmic negative tumors was 9.35 times (P value < 0.0001) that compared with patients with cytoplasmic positive tumors after adjusting for ER, PR, and HER2 status. Cytoplasmic cyclin E expression also predicts long-term outcome and is associated with reduced disease free, recurrence free, and overall survival rates, independent of increased pretreatment tumor infiltrating lymphocytes. CONCLUSIONS: Cyclin E independently predicted response to neoadjuvant chemotherapy. Hence, its routine immunohistochemical analysis could be used clinically to identify those breast cancer patients expected to have a poor response to anthracycline/taxane-based chemotherapy.

Enhancer transcription reveals subtype-specific gene expression programs controlling breast cancer pathogenesis.

Noncoding transcription is a defining feature of active enhancers, linking transcription factor (TF) binding to the molecular mechanisms controlling gene expression. To determine the relationship between enhancer activity and biological outcomes in breast cancers, we profiled the transcriptomes (using GRO-seq and RNA-seq) and epigenomes (using ChIP-seq) of 11 different human breast cancer cell lines representing five major molecular subtypes of breast cancer, as well as two immortalized ("normal") human breast cell lines. In addition, we developed a robust and unbiased computational pipeline that simultaneously identifies putative subtype-specific enhancers and their cognate TFs by integrating the magnitude of enhancer transcription, TF mRNA expression levels, TF motif P-values, and enrichment of H3K4me1 and H3K27ac. When applied across the 13 different cell lines noted above, the Total Functional Score of Enhancer Elements (TFSEE) identified key breast cancer subtype-specific TFs that act at transcribed enhancers to dictate gene expression patterns determining growth outcomes, including Forkhead TFs, FOSL1, and PLAG1. FOSL1, a Fos family TF, (1) is highly enriched at the enhancers of triple negative breast cancer (TNBC) cells, (2) acts as a key regulator of the proliferation and viability of TNBC cells, but not Luminal A cells, and (3) is associated with a poor prognosis in TNBC breast cancer patients. Taken together, our results validate our enhancer identification pipeline and reveal that enhancers transcribed in breast cancer cells direct critical gene regulatory networks that promote pathogenesis.

Specific, reversible G1 arrest by UCN-01 in vivo provides cytostatic protection of normal cells against cytotoxic chemotherapy in breast cancer.

BACKGROUND: Low-dose UCN-01 mediates G1 arrest in normal proliferating cell lines with an intact G1 to S transition but not tumour cells with a deregulated G1 to S checkpoint. Here we hypothesised that UCN-01 is effective in mediating a selective, reversible G1 arrest of normal proliferating cells, resulting in decreased chemotoxicity, improved tolerance and enhanced chemotherapeutic efficacy in vivo in both non-tumour-bearing mice and in breast cancer cell line xenograft models. METHODS: Murine small bowel epithelium was used to examine the kinetics and mechanism of low-dose UCN-01-mediated arrest of normal proliferating cells and if it can protect tumour-bearing mice (MDA-MB-468 xenografts) against the toxic effects of chemotherapy (5-fluorouricil (5-FU)) allowing for its full therapeutic activity. RESULTS: UCN-01 causes significant, reversible arrest of normal gut epithelial cells at 24 h; this arrest persists for up to 7 days. Normal cellular proliferation returns by 2 weeks. Pre-treatment of both non-tumour-bearing and MDA-MB-468 tumour-bearing mice with UCN-01 prior to bolus 5-FU (450 mg/kg) yielded enhanced therapeutic efficacy with significantly decreased tumour volumes and increased survival. CONCLUSIONS: UCN-01 mediates a specific, reversible G1 arrest of normal cells in vivo and provides a cytoprotective strategy that decreases toxicity of cytotoxic chemotherapy without compromising efficacy.

Histone modification profiling in breast cancer cell lines highlights commonalities and differences among subtypes.

BACKGROUND: Epigenetic regulators are frequently mutated or aberrantly expressed in a variety of cancers, leading to altered transcription states that result in changes in cell identity, behavior, and response to therapy. RESULTS: To define alterations in epigenetic landscapes in breast cancers, we profiled the distributions of 8 key histone modifications by ChIP-Seq, as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes, across 13 distinct cell lines that represent 5 molecular subtypes of breast cancer and immortalized human mammary epithelial cells. DISCUSSION: Using combinatorial patterns of distinct histone modification signals, we defined subtype-specific chromatin signatures to nominate potential biomarkers. This approach identified AFAP1-AS1 as a triple negative breast cancer-specific gene associated with cell proliferation and epithelial-mesenchymal-transition. In addition, our chromatin mapping data in basal TNBC cell lines are consistent with gene expression patterns in TCGA that indicate decreased activity of the androgen receptor pathway but increased activity of the vitamin D biosynthesis pathway. CONCLUSIONS: Together, these datasets provide a comprehensive resource for histone modification profiles that define epigenetic landscapes and reveal key chromatin signatures in breast cancer cell line subtypes with potential to identify novel and actionable targets for treatment.

Cytoplasmic Cyclin E and Phospho-Cyclin-Dependent Kinase 2 Are Biomarkers of Aggressive Breast Cancer.

Cyclin E and its co-activator, phospho-cyclin-dependent kinase 2 (p-CDK2), regulate G1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated full-length from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer.

How will we recruit, train, and retain physicians and scientists to conduct translational cancer research?

Advances in clinical medicine require effective translational research. Ideally, this research will be performed by multidisciplinary teams that include both physicians and basic scientists. However, the current system does not appropriately train either physicians or basic scientists for these careers. In addition, translational researchers are often not properly rewarded, and this creates a disincentive for pursuing this kind of research. The roles and challenges for physicians and basic researchers in the field of translational research are discussed along with proposed solutions for improving their recruitment, training, and retention. Cancer 2015;121:806-816. © 2014 American Cancer Society.

A phase 1 study with dose expansion of the CDK inhibitor dinaciclib (SCH 727965) in combination with epirubicin in patients with metastatic triple negative breast cancer.

PURPOSE: Low molecular weight cyclin E (LMW-E) isoforms, overexpressed in a majority (~70 %) of triple-negative breast cancers (TNBC), were found in preclinical models to mediate tumorigenesis through binding and activation of CDK2. CDK1/CDK2 inhibitors, such as dinaciclib, combined with anthracyclines, were synergistic in decreasing viability of TNBC cell lines. Based on this data, a phase 1 study was conducted to determine the maximum tolerated dose of dinaciclib in combination with epirubicin in patients with metastatic TNBC. METHODS: Cohorts of at least 2 patients were treated with escalating doses of dinaciclib given on day 1 followed by standard dose of epirubicin given on day 2 of a 21 day cycle. No intra-patient dose escalation was allowed. An adaptive accrual design based upon toxicity during cycle 1 determined entry into therapy cohorts. The target acceptable dose limiting toxicity (DLT) to advance to the next treatment level was 30 %. RESULTS: Between 9/18/2012 and 7/18/2013, 9 patients were enrolled and treated at MD Anderson Cancer Center. DLTs included febrile neutropenia (grade 3, n = 2), syncope (grade 3, n = 2) and vomiting (grade 3, n = 1). Dose escalation did not proceed past the second cohort due to toxicity. After further accrual, the first dose level was also found to be too toxic. No treatment responses were noted, median time to progression was 5.5 weeks (range 3-12 weeks). Thus, accrual was stopped rather than explore the -1 dose level. CONCLUSION: The combination of dinaciclib and epirubicin is associated with substantial toxicities and does not appear to be an effective treatment option for TNBC.

LMW-E/CDK2 deregulates acinar morphogenesis, induces tumorigenesis, and associates with the activated b-Raf-ERK1/2-mTOR pathway in breast cancer patients.

Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2-associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E-expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2-associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib.

Elafin is downregulated during breast and ovarian tumorigenesis but its residual expression predicts recurrence.

INTRODUCTION: Elafin is an endogenous serine protease inhibitor. The majority of breast cancer cell lines lack elafin expression compared to human mammary epithelial cells. In this study, we hypothesized that elafin is downregulated during breast and ovarian tumorigenesis. METHODS: We examined elafin expression by immunohistochemistry (IHC) in specimens of normal breast tissue (n = 24), ductal carcinoma in situ (DCIS) (n = 54), and invasive breast cancer (n = 793). IHC analysis of elafin expression was also performed in normal fallopian tube tissue (n = 20), ovarian cystadenomas (n = 9), borderline ovarian tumors (n = 21), and invasive ovarian carcinomas (n = 216). To understand the significance of elafin in luminal breast cancer cell lines, wild-type or M25G elafin (lacking the protease inhibitory function) were exogenously expressed in MCF-7 and T47D cells. RESULTS: Elafin expression was downregulated in 24% of DCIS and 83% of invasive breast tumors when compared to elafin expression in the normal mammary epithelium. However, the presence of elafin-positive cells in invasive breast tumors, even at low frequency, correlated with poor recurrence-free survival (RFS), reduced overall survival (OS), and clinicopathological markers of aggressive tumor behavior. Elafin-positive cells were an especially strong and independent prognostic marker of reduced RFS in IHC-defined luminal A-like tumors. Elafin was also downregulated in 33% of ovarian cystadenomas, 43% of borderline ovarian tumors, and 86% of invasive ovarian carcinomas when compared to elafin expression in the normal fallopian tube. In ovarian tumors, elafin-positive cells were correlated with reduced RFS, OS and disease-specific survival (DSS) only in stage I/II patients and not in stage III/IV patients. Notably, exogenous expression of elafin or elafin M25G in the luminal breast cancer cell lines MCF-7 and T47D significantly decreased cell proliferation in a protease inhibitory domain-independent manner. CONCLUSIONS: Elafin predicts poor outcome in breast and ovarian cancer patients and delineates a subset of endocrine receptor-positive breast cancer patients susceptible to recurrence who could benefit from more aggressive intervention. Our in vitro results suggest that elafin arrests luminal breast cancer cells, perhaps suggesting a role in tumor dormancy.

Exploring Novel Frontiers: Leveraging STAT3 Signaling for Advanced Cancer Therapeutics.

Signal Transducer and Activator of Transcription 3 (STAT3) plays a significant role in diverse physiologic processes, including cell proliferation, differentiation, angiogenesis, and survival. STAT3 activation via phosphorylation of tyrosine and serine residues is a complex and tightly regulated process initiated by upstream signaling pathways with ligand binding to receptor and non-receptor-linked kinases. Through downstream deregulation of target genes, aberrations in STAT3 activation are implicated in tumorigenesis, metastasis, and recurrence in multiple cancers. While there have been extensive efforts to develop direct and indirect STAT3 inhibitors using novel drugs as a therapeutic strategy, direct clinical application remains in evolution. In this review, we outline the mechanisms of STAT3 activation, the resulting downstream effects in physiologic and malignant settings, and therapeutic strategies for targeting STAT3. We also summarize the pre-clinical and clinical evidence of novel drug therapies targeting STAT3 and discuss the challenges of establishing their therapeutic efficacy in the current clinical landscape.

Neutrophil Elastase Remodels Mammary Tumors to Facilitate Lung Metastasis.

Metastatic disease remains the leading cause of death due to cancer, yet the mechanism(s) of metastasis and its timely detection remain to be elucidated. Neutrophil elastase (NE), a serine protease secreted by neutrophils, is a crucial mediator of chronic inflammation and tumor progression. In this study, we used the PyMT model (NE+/+ and NE-/-) of breast cancer to interrogate the tumor-intrinsic and -extrinsic mechanisms by which NE can promote metastasis. Our results showed that genetic ablation of NE significantly reduced lung metastasis and improved metastasis-free survival. RNA-sequencing analysis of primary tumors indicated differential regulation of tumor-intrinsic actin cytoskeleton signaling pathways by NE. These NE-regulated pathways are critical for cell-to-cell contact and motility and consistent with the delay in metastasis in NE-/- mice. To evaluate whether pharmacologic inhibition of NE inhibited pulmonary metastasis and phenotypically mimicked PyMT NE-/- mice, we utilized AZD9668, a clinically available and specific NE inhibitor. We found AZD9668 treated PyMT-NE+/+ mice showed significantly reduced lung metastases, improved recurrence-free, metastasis-free and overall survival, and their tumors showed similar molecular alterations as those observed in PyMT-NE-/- tumors. Finally, we identified a NE-specific signature that predicts recurrence and metastasis in patients with breast cancer. Collectively, our studies suggest that genetic ablation and pharmacologic inhibition of NE reduces metastasis and extends survival of mouse models of breast cancer, providing rationale to examine NE inhibitors as a treatment strategy for the clinical management of patients with metastatic breast cancer.

Cell cycle arrest induces lipid droplet formation and confers ferroptosis resistance.

How cells coordinate cell cycling with cell survival and death remains incompletely understood. Here, we show that cell cycle arrest has a potent suppressive effect on ferroptosis, a form of regulated cell death induced by overwhelming lipid peroxidation at cellular membranes. Mechanistically, cell cycle arrest induces diacylglycerol acyltransferase (DGAT)-dependent lipid droplet formation to sequester excessive polyunsaturated fatty acids (PUFAs) that accumulate in arrested cells in triacylglycerols (TAGs), resulting in ferroptosis suppression. Consequently, DGAT inhibition orchestrates a reshuffling of PUFAs from TAGs to phospholipids and re-sensitizes arrested cells to ferroptosis. We show that some slow-cycling antimitotic drug-resistant cancer cells, such as 5-fluorouracil-resistant cells, have accumulation of lipid droplets and that combined treatment with ferroptosis inducers and DGAT inhibitors effectively suppresses the growth of 5-fluorouracil-resistant tumors by inducing ferroptosis. Together, these results reveal a role for cell cycle arrest in driving ferroptosis resistance and suggest a ferroptosis-inducing therapeutic strategy to target slow-cycling therapy-resistant cancers.

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.

Staurosporine is chemoprotective by inducing G1 arrest in a Chk1- and pRb-dependent manner.

Chemotherapeutic agents have been the mainstay of cancer therapy for years. However, their effectiveness has been limited by toxicities they impart on normal cells. Staurosporine (ST) has been shown to arrest normal, but not breast cancer, cells in G1. Therefore, ST may become a chemoprotective agent, arresting normal cells while allowing tumor cells to enter cell cycle phases where they are sensitive to chemotherapeutic agents. Understanding the mechanism of ST-mediated G1 arrest may allow for a beneficial chemoprotective treatment strategy for patients. We utilized 76NE6 (pRb+/p53-), 76NF2V (pRb+/p53+) and 76NE7 (pRb-/P53+) non-tumorigenic human mammary epithelial cell lines to understand the role of the Rb and p53 pathways in ST-directed G1 arrest. CDK4 was downregulated by ST in Rb+ cells, but its presence could not reverse the arrest, neither did its stable downregulation alter ST-mediated cellular response. ST-mediated G1 arrest required pRb, which in turn initiated a cascade of events leading to inhibition of CDK4. Further assessment of this pathway revealed that Chk1 expression and activity were required for the Rb-dependent arrest. For example, pRb+ cells with small interfering RNA to Chk1 had approximately 60% less cells in G1 phase compared with controls and pRb- cells do not arrest upon ST. Furthermore, Chk1 expression facilitates the release of the Rb+ cells from G1 arrest. Collectively, our data suggest that pRb cooperates with Chk1 to mediate a G1 arrest only in pRb+ cells. The elucidation of this pathway can help identify novel agents to protect cancer patients against the debilitating effects of chemotherapy.

Elafin, an inhibitor of elastase, is a prognostic indicator in breast cancer.

INTRODUCTION: Elafin is an elastase-specific inhibitor with increased transcription in normal mammary epithelial cells compared to mammary carcinoma cells. In this report, we test the hypothesis that inhibition of elastase, through induction of elafin, leads to inhibition of human breast cancer cell viability and, therefore, predicts survival in breast cancer patients. METHODS: Panels of normal and immortalized breast epithelial cells, along with breast carcinoma cells, were used to examine the impact of adenoviral-mediated elafin expression or shRNA-mediated inhibition of elastase on the growth of cells and xenografts in nude mice. To determine the prognostic significance of decreased elafin in patients with invasive breast cancer, previously published gene array datasets were interrogated. RESULTS: Elafin expression had no effect on non-tumorigenic cells but resulted in marked inhibition of cell growth in breast cancer cell lines. Control-treated xenografts generated a tumor burden that necessitated sacrifice within one month of initial treatment, whereas xenograft-bearing mice treated with Ad-Elafin were alive at eight months with marked reduction in tumor growth. Elastase inhibition mimicked these results, showing decreased tumor cell growth in vitro and in vivo. Low expression of elafin gene correlated with significantly reduced time to relapse, and when combined with high expression of elastase gene was associated with decreased survival in breast cancer patients. CONCLUSION: Our data suggest that elafin plays a direct role in the suppression of tumors through inhibition of elastase and thus serves as a prognostic indicator for breast cancer patients.

Distinct Mechanisms of Resistance to CDK4/6 Inhibitors Require Specific Subsequent Treatment Strategies: One Size Does Not Fit All.

Cyclin-dependent kinase (CDK) 4/6 inhibitors have transformed the treatment landscape of patients with hormone receptor-positive breast cancers. However, despite improvements in clinical outcomes, the approximately 70% of patients with tumors that are not intrinsically resistant to a CDK4/6 inhibitor still ultimately acquire resistance, which leads to a dilemma for clinicians when deciding which treatment to offer patients when they demonstrate disease progression on a CDK4/6 inhibitor. As such, many groups have sought to understand the mechanisms of resistance to CDK4/6 inhibitors, mostly focusing on genetic alterations associated with resistance. Though several recurrent mutations have been described, they are not consistent enough to guide clinical practice or generate novel rational treatment options. Two recent publications have used transcriptomic analysis to unravel distinct mechanisms driving resistance to individual CDK4/6 inhibitors and in doing so have identified biomarkers that could potentially help identify the next course of treatment for patients following disease progression.

Sequential Targeting of Retinoblastoma and DNA Synthesis Pathways Is a Therapeutic Strategy for Sarcomas That Can Be Monitored in Real Time.

Treatment strategies with a strong scientific rationale based on specific biomarkers are needed to improve outcomes in patients with advanced sarcomas. Suppression of cell-cycle progression through reactivation of the tumor suppressor retinoblastoma (Rb) using CDK4/6 inhibitors is a potential avenue for novel targeted therapies in sarcomas that harbor intact Rb signaling. Here, we evaluated combination treatment strategies (sequential and concomitant) with the CDK4/6 inhibitor abemacicib to identify optimal combination strategies. Expression of Rb was examined in 1,043 sarcoma tumor specimens, and 50% were found to be Rb-positive. Using in vitro and in vivo models, an effective two-step sequential combination strategy was developed. Abemaciclib was used first to prime Rb-positive sarcoma cells to reversibly arrest in G1 phase. Upon drug removal, cells synchronously traversed to S phase, where a second treatment with S-phase targeted agents (gemcitabine or Wee1 kinase inhibitor) mediated a synergistic response by inducing DNA damage. The response to treatment could be noninvasively monitored using real-time positron emission tomography imaging and serum thymidine kinase activity. Collectively, these results show that a novel, sequential treatment strategy with a CDK4/6 inhibitor followed by a DNA-damaging agent was effective, resulting in synergistic tumor cell killing. This approach can be readily translated into a clinical trial with noninvasive functional imaging and serum biomarkers as indicators of response and cell cycling. SIGNIFICANCE: An innovative sequential therapeutic strategy targeting Rb, followed by treatment with agents that perturb DNA synthesis pathways, results in synergistic killing of Rb-positive sarcomas that can be noninvasively monitored.

Abemaciclib Is Effective in Palbociclib-Resistant Hormone Receptor-Positive Metastatic Breast Cancers.

Cyclin-dependent kinases 4/6 inhibitor (CDK4/6i) plus endocrine therapy (ET) is standard of care for patients with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (MBC). However, resistance to CDK4/6is plus ET remains a clinical problem with limited therapeutic options following disease progression. Different CDK4/6is might have distinct mechanisms of resistance, and therefore using them sequentially or targeting their differentially altered pathways could delay disease progression. To understand pathways leading to resistance to the CDK4/6is palbociclib and abemaciclib, we generated multiple in vitro models of palbociclib-resistant (PR) and abemaciclib-resistant (AR) cell lines as well as in vivo patient-derived xenografts (PDX) and ex vivo PDX-derived organoids (PDxO) from patients who progressed on CDK4/6i. PR and AR breast cancer cells exhibited distinct transcriptomic and proteomic profiles that sensitized them to different classes of inhibitors; PR cells upregulated G2-M pathways and responded to abemaciclib, while AR cells upregulated mediators of the oxidative phosphorylation pathway (OXPHOS) and responded to OXPHOS inhibitors. PDX and organoid models derived from patients with PR breast cancer remained responsive to abemaciclib. Resistance to palbociclib while maintaining sensitivity to abemaciclib was associated with pathway-specific transcriptional activity but was not associated with any individual genetic alterations. Finally, data from a cohort of 52 patients indicated that patients with HR-positive/HER2-negative MBC who progressed on palbociclib-containing regimens can exhibit a meaningful overall clinical benefit from abemaciclib-based therapy when administered after palbociclib. These findings provide the rationale for clinical trials evaluating the benefit of abemaciclib treatment following progression on a prior CDK4/6i. SIGNIFICANCE: Palbociclib-resistant breast cancers respond to abemaciclib and express pathway-specific signatures of sensitivity, providing a biomarker-driven therapeutic option for patients with metastatic breast cancer following disease progression on cyclin-dependent kinases 4/6 inhibitors.

Adavosertib Enhances Antitumor Activity of Trastuzumab Deruxtecan in HER2-Expressing Cancers.

PURPOSE: Cyclin E (CCNE1) has been proposed as a biomarker of sensitivity to adavosertib, a Wee1 kinase inhibitor, and a mechanism of resistance to HER2-targeted therapy. EXPERIMENTAL DESIGN: Copy number and genomic sequencing data from The Cancer Genome Atlas and MD Anderson Cancer Center databases were analyzed to assess ERBB2 and CCNE1 expression. Molecular characteristics of tumors and patient-derived xenografts (PDX) were assessed by next-generation sequencing, whole-exome sequencing, fluorescent in situ hybridization, and IHC. In vitro, CCNE1 was overexpressed or knocked down in HER2+ cell lines to evaluate drug combination efficacy. In vivo, NSG mice bearing PDXs were subjected to combinatorial therapy with various treatment regimens, followed by tumor growth assessment. Pharmacodynamic markers in PDXs were characterized by IHC and reverse-phase protein array. RESULTS: Among several ERBB2-amplified cancers, CCNE1 co-amplification was identified (gastric 37%, endometroid 43%, and ovarian serous adenocarcinoma 41%). We hypothesized that adavosertib may enhance activity of HER2 antibody-drug conjugate trastuzumab deruxtecan (T-DXd). In vitro, sensitivity to T-DXd was decreased by cyclin E overexpression and increased by knockdown, and adavosertib was synergistic with topoisomerase I inhibitor DXd. In vivo, the T-DXd + adavosertib combination significantly increased γH2AX and antitumor activity in HER2 low, cyclin E amplified gastroesophageal cancer PDX models and prolonged event-free survival (EFS) in a HER2-overexpressing gastroesophageal cancer model. T-DXd + adavosertib treatment also increased EFS in other HER2-expressing tumor types, including a T-DXd-treated colon cancer model. CONCLUSIONS: We provide rationale for combining T-DXd with adavosertib in HER2-expressing cancers, especially with co-occuring CCNE1 amplifications. See related commentary by Rolfo et al., p. 4317.