CDK4/6 Inhibitors Research Articles

Real-World Treatment Patterns and Clinical Outcomes among Patients Receiving CDK4/6 Inhibitors for Metastatic Breast Cancer in a Canadian Setting Using AI-Extracted Data

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are widely used in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 negative (HER2−) advanced/metastatic breast cancer (ABC/MBC) in first line (1L), but little is known about their real-world use and clinical outcomes long-term, in Canada. This study used Pentavere’s previously validated artificial intelligence (AI) to extract real-world data on the treatment patterns and outcomes of patients receiving CDK4/6i+endocrine therapy (ET) for HR+/HER2− ABC/MBC at Sinai Health in Toronto, Canada. Between 1 January 2016 and 1 July 2021, 48 patients were diagnosed with HR+/HER2− ABC/MBC and received CDK4/6i + ET. A total of 38 out of 48 patients received CDK4/6i + ET in 1L, of which 34 of the 38 (89.5%) received palbociclib + ET. In 2L, 12 of the 21 (57.1%) patients received CDK4/6i + ET, of which 58.3% received abemaciclib. In 3L, most patients received chemotherapy (10/12, 83.3%). For the patients receiving CDK4/6i in 1L, the median (95% CI) time to the next treatment was 42.3 (41.2, NA) months. The median (95% CI) time to chemotherapy was 46.5 (41.4, NA) months. The two-year overall survival (95% CI) was 97.4% (92.4, 100.0), and the median (range) follow-up was 28.7 (3.4–67.6) months. Despite the limitations inherent in real-world studies and a limited number of patients, these AI-extracted data complement previous studies, demonstrating the effectiveness of CDK4/6i + ET in the Canadian real-world 1L, with most patients receiving palbociclib as CDK4/6i in 1L.

Abstract CT074: ELECTRA: An open-label, multicenter, phase 1b/2 study of elacestrant in combination with abemaciclib in patients with brain metastasis from estrogen receptor-positive (ER+), HER2-negative (HER2-) breast cancer (BC)

Abstract Background: Endocrine therapy (ET) + cyclin dependent kinase 4/6 inhibitor (CDK4/6i) is the mainstay for the management of ER+/HER2- mBC as 1st-line therapy. However, tumors eventually develop resistance to ET, leading to disease progression. In the EMERALD phase 3 trial, single-agent elacestrant was associated with significantly prolonged progression-free survival (PFS) and a manageable safety profile vs standard of care (SOC) ET, leading to the first oral SERD approval. Patients receiving elacestrant experienced a median PFS (mPFS) of 3.8 months vs 1.9 months with SOC (Bidard, 2022). Patients who received ≥12 months of prior CDK4/6i experienced a mPFS of 8.6 months with elacestrant vs 1.9 months with SOC (SABCS, 2022). Currently, there are no approved systemic treatments for patients with ER+/HER2- BC who have brain metastasis. The rationale for the ELECTRA study is to combine elacestrant with abemaciclib in patients with brain metastasis, given both compounds have demonstrated the ability to cross the blood-brain barrier (Conlan, 2020; Tolaney, 2020). Methods: ELECTRA (NCT05386108) is an open-label phase 1b/2, multicenter study evaluating elacestrant in combination with abemaciclib. Eligibility includes patients with ER+/HER2- locally advanced or metastatic BC and measurable brain metastasis. For phase 1b, the presence of brain metastasis is not required for eligibility. For phase 2, patients must have ≥ 1 active and measurable brain metastasis per RECIST v1.1. Patients must have received prior therapy in the metastatic setting, including ≥ 1 endocrine therapy, ≤ 2 chemotherapy regimens, and 0-2 prior CDK4/6i (excluding abemaciclib). The phase 1b primary objective is to determine the recommended phase 2 dose (RP2D); secondary objectives include safety, pharmacokinetics (PK), overall response rate (ORR), duration of response (DoR), clinical benefit rate (CBR), PFS, and overall survival (OS). The phase 2 primary objective is ORR per RECIST v1.1; secondary objectives include intracranial response rate, DoR, CBR, PFS, OS, PK, and quality of life. The phase 1b portion of the trial is actively ongoing to determine the RP2D with study sites open worldwide. Citation Format: Erika Hamilton, Sung-Bae Kim, Nancy U. Lin, Ahmad Awada, Eva Ciruelos Gil, Alessandro Di Sanzo, Bartomeu Piza Vallespir, Kathy Puyana Theall, Nuhad Ibrahim. ELECTRA: An open-label, multicenter, phase 1b/2 study of elacestrant in combination with abemaciclib in patients with brain metastasis from estrogen receptor-positive (ER+), HER2-negative (HER2-) breast cancer (BC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT074.

Therapy for Hormone Receptor-Positive, Human Epidermal Growth Receptor 2-Negative Metastatic Breast Cancer Following Treatment Progression via CDK4/6 Inhibitors: A Literature Review.

Endocrine therapy (ET) with a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) is currently the first-line standard treatment for most patients with hormone receptor-positive (HR+) and human epidermal growth receptor 2-negative (HER2-) metastatic or advanced breast cancer. However, the majority of tumors response to and eventually develop resistance to CDK4/6is. The mechanisms of resistance are poorly understood, and the optimal postprogression treatment regimens and their sequences continue to evolve in the rapidly changing treatment landscape. In this review, we generally summarize the mechanisms of resistance to CDK4/6is and ET, and describe the findings from clinical trials using small molecule inhibitors, antibody-drug conjugates and immunotherapy, providing insights into how these novel strategies may reverse treatment resistance, and discussing how some have not translated into clinical benefit. Finally, we provide rational treatment strategies based on the current emerging evidence.

Selective CDK7 inhibition suppresses cell cycle progression and MYC signaling while enhancing apoptosis in therapy-resistant estrogen receptor positive breast cancer.

Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER) positive (ER+) breast cancer (BC). Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine resistant ER+ BC models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ BC. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ BC remain elusive. Herein, we sought to unravel these mechanisms. We conducted multi-omic analyses in ER+ BC models in vitro and in vivo including models with different genetic backgrounds. We also performed genome wide CRISPR knock-out library screens to identify potential therapeutic vulnerabilities in CDK4/6i resistance models. We found that the on-target anti-tumor effects of CDK7 inhibition in ER+ BC are in part p53 dependent, involve cell-cycle inhibition and suppression of c-MYC. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ETs and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118, however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Lastly, genome wide CRISPR/Cas9 screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i resistant models. Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ BC. In addition, our study highlights the potential of increased MYC activity and intact P53 as predictors for sensitivity to CDK7 inhibitor-based treatments.

Abstract 6253: Single-cell RNA sequencing reveals metastatic site-specific biomarkers for predicting response to CDK4/6 inhibition in advanced HR+/HER2- breast cancer

Abstract Background: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), in combination with endocrine therapy, are established as standard treatment for advanced HR+/HER2- breast cancer (BC). However, response heterogeneity is observed, with approximately one-third of patients exhibiting intrinsic resistance. This study investigates the mechanisms of variable responses to CDK4/6i through single-cell RNA sequencing of metastatic site biopsies, revealing distinct biomarkers for predicting CDK4/6i response. Methods: Single-cell RNA sequencing analyzed metastatic tumors from advanced HR+/HER2- BC patients pre-CDK4/6i treatment at baseline (BL) and/or at disease progression. BL samples were from CDK4/6i responders (n = 8, median progression-free survival [mPFS] = 17 months), while progressors were categorized as early-progressors (EP, n = 3, mPFS = 3 months) and late-progressors (LP, n = 8, mPFS = 10 months). Metastatic sites included liver (10), pleural effusions (6), ascites (2), and bone (1). InferCNV distinguished tumor cells, and functional analysis utilized the Molecular Signatures Database. Results: Both EP and LP tumor cells exhibited reduced estrogen response and increased IFN-γ pathways compared to BL tumors. LP tumors displayed enhanced Myc targets, EMT, TNF-α, and inflammatory pathways compared to EP tumors, revealing differential mechanisms of early versus late progression. Predictive signatures, including TFF3 and MGP, were significantly upregulated in BL tumor cells across different metastatic sites. Metastasis-specific markers included APOA and SERPINA1 (liver), and AGR2 (pleural effusion). BL and LP responders showed increased tumor infiltrating CD8+ T cell and NK cells compared to EP non-responders. Notably, despite a high CD8+ T frequency in responder tumors, functional analysis revealed a significant upregulation of genes associated with stress/apoptosis and exhaustion in proliferative CD4+ and CD8+ T cells from BL compared to LP tumors, including HSP90 and HSPA8, associated with poor response to immune checkpoint inhibitors. These findings suggest potential benefits of immunotherapy for CDK4/6i late progressors. Longitudinal biopsies further revealed dynamic NK cell expansion and enhanced cytotoxic T cell activity alongside the downregulation of stress/apoptosis gene expression at progression compared to BL tumors. Conclusion: This study underscores the significance of metastatic site-specific biomarkers in predicting outcomes to CDK4/6i. Tumor-infiltration lymphocytes may also serve as baseline predictors, while stress/apoptosis gene signatures suggest checkpoint inhibitor usage in CDK4/6i late progressors. These potentially actionable findings emphasize the need to optimize therapeutic approaches based on microenvironment-specific changes following disease progression. Citation Format: Linjie Luo, Peng Yang, Sofia Mastoraki, Yan Wang, Nicole M. Kettner, Akshara Singareeka Raghavendra, Debasish Tripathy, Senthil Damodaran, Kelly K. Hunt, Ziyi Li, Khandan Keyomarsi. Single-cell RNA sequencing reveals metastatic site-specific biomarkers for predicting response to CDK4/6 inhibition in advanced HR+/HER2- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6253.

Abstract 5719: Targeting CDK4/6 and BET proteins for the treatment of RB+ and RB- osteosarcoma

Abstract Osteosarcoma (OS) is the most common malignant bone cancer characterized by heterogeneity and genomic instability. Treatment and survival of patients have not improved in several decades, making it urgent to develop safe and effective therapies. Dysregulation of cell cycle regulators CDKN2A, cyclins, and CDK4/6 represent high-risk signatures in aggressive OS and can be targeted via CDK4/6 inhibitors (CDK4/6i). However, monotherapy often fails due to resistance from speculated RB loss as well as hyperactivation of compensatory signaling pathways in RB proficient cancers. In addition, >70% of OS patients have RB loss which is viewed as a biomarker of therapeutic response that negates the use of CDK4/6i. However, more research is needed in RB-deficient preclinical models to clearly define and validate the utility of RB as a biomarker for patient stratification. Based on our screening data, we hypothesize that combining CDK4/6i with bromodomain and extraterminal domain inhibitors (BETi) will potentiate anti-tumorigenic effects regardless of RB status in OS. BETi creates imbalances between transcription and replication which worsens oncogenic stress. In OS cell lines, combination index and Bliss analysis indicated additive-to-synergistic growth inhibition using CDK4/6i (abemaciclib or palbociclib) and BETi (AZD5153). Dual therapy also resulted in increased apoptosis at clinically relevant concentrations in an OS xenoline (TT2) compared to monotherapy. To determine potential for emergence of therapeutic resistance, an in-vitro longitudinal growth delay assay was established. While single agent therapies resulted in transient decreases in cell numbers over time, combination CDK4/6i+BETi resulted in cell death (p<0.05). To determine the effect of RB loss on therapeutic response to CDK4/6i, CRISPR mediated RB-knockout (KO) clones were developed in 143B human and K7M2 murine OS cell lines. RB KO cells exhibited sensitivity to CDK4/6i irrespective of RB status. However, levels of CDK4/6i required for 50% growth inhibition in RB KO were 1-3-fold higher using palbociclib, with negligible differences observed with abemaciclib in RB KO vs WT clones. To determine if therapeutic response is achievable in-vivo, a single-agent dosing study was conducted using palbociclib (40mg/kg, SID) and AZD5153 (1mg/kg, BID) in patient-derived xenograft (PDX) model derived from a metastatic lesion in the pelvis (TT2 PDX) harboring a CDK4/6 hyperactivation signature and monoallelic RB loss. Tumor growth reduction and survival was significantly higher in monotherapy groups compared to vehicle. However, one week after dosing was stopped, survival rates based on tumor volume endpoint were 40% and 100% for palbociclib and AZD5153 respectively. In-vivo efficacy of dual CDK46i+BETi is underway. This data provides rationale for further study of novel therapeutic options which may expand the clinical utility of CDK4/6i regardless of RB status. Citation Format: Rada Malko, Pankita H. Pandya, Harlan E. Shannon, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Farinaz Barghi, Nikan Riyahi, Ryli Justice, Melissa A. Trowbridge, Kathryn L. Coy, Anthony L. Sinn, Khadijeh Bijangi-Vishehsaraei, Micheal J. Ferguson, M. Reza Saadatzadeh, Karen E. Pollok. Targeting CDK4/6 and BET proteins for the treatment of RB+ and RB- osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5719.

Abstract 526: Identification of CDK6 binding proteins by proximity labeling reveals novel mechanisms for CDK4/6 inhibitor resistance in breast cancer

Abstract Breast cancer is one of the most prevalent cancer type in human, and the majority (~70%) is the hormone positive (HR+) subtype. The current frontline treatment of choice in metastatic HR(+) breast cancer is CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy. Current guidelines recommend CDK4/6i treatment as 1st or 2nd line, as CDK4/6i provides survival benefit and good quality of life. However, resistance eventually emerges, leading to discontinuation of CDK4/6i and switching to subsequent treatment. Investigation of mechanisms to CDK4/6i resistance is an active area of intense research. CDK6 overexpression has been reported as an important mechanism for CDK4/6i resistance by several independent groups. We incidentally discovered a point mutation in the CDK6 protein that abolishes the CDK4/6i resistance conferred by CDK6 overexpression. When treated with CDK4/6i, overexpression of wild type CDK6 did not suppress G1/S, while both non-transfected and overexpression of mutant CDK6 both had similar suppression of G1/S. We hypothesized that a binding partner to CDK6 demonstrated differential binding activity to the mutant which abolished the resistance by CDK6 overexpression. To assess this hypothesis, we performed proximity labeling (PL) by APEX2. Both wild type and mutant CDK6 were cloned into APEX-Flag/V5 vectors with either cytoplasm (NES) or nuclear (NLS) signals. Mass spectrometry following treatment by H2O2 induced biotinylation produced candidate peptides that bound with overexpressed CDK6. We identified leading candidates of differentially binding partners that bound to mutant CDK6. Further experimental validation was performed to confirm physical proximity between CDK6 and the candidate protein. Our research uncovered novel insights to how CDK6 conferred resistance towards CDK4/6i and expands knowledge into further therapeutic development. Citation Format: Chung Wei Ting, Yong-Ji Zhuang, Chih-Yi Lin, Chung-Jen Yu, Ta-Chung Chao, Ling-Ming Tseng, Chun-Yu Liu, Yi-Fang Tsai, Chi-Cheng Huang, Jiun-I Lai. Identification of CDK6 binding proteins by proximity labeling reveals novel mechanisms for CDK4/6 inhibitor resistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 526.

Abstract 4622: Anti-tumor activity of CDK2 inhibitor BLU-222 in combination with CDK4/6 inhibitors for overcoming resistance in HR positive and triple negative metastatic breast cancers models

Abstract Background: Cyclin-dependent-kinase-4/6 inhibitors (CDK4/6is) plus endocrine therapy (ET) are standard of care first-line treatment for patients with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (mBC). However, the emergence of resistance to CDK4/6is plus ET presents a clinical challenge with few treatment alternatives. The effectiveness of CDK4/6is in patients with triple-negative (TN) breast cancer remains uncertain, although the potential synergy with other targeted therapies is currently under investigation. Our study demonstrates the enhanced and synergistic activity of BLU-222, a selective CDK2 inhibitor, when combined with CDK4/6is in preclinical models of both HR+/HER2- and TN breast cancer resistant to CDK4/6is. Methods: Palbociclib resistant (PR) HR+/HER2- (MCF7 and T47D) and TN (MDA-MB-231 and BT-20) breast cancer cell lines were generated by escalating palbociclib concentrations in culture. Using SynergyFinder, we assessed the effect of BLU-222 alone and in combination with palbociclib in the highest single-agent model in vitro. The effectiveness of BLU-222, alone or in combination with palbociclib, was further evaluated in four patient-derived xenograft (PDX) models from HR+/HER2- patients whose tumors progressed after palbociclib plus ET, two TN PDX models, and a TN breast cancer transgenic model driven by tumor-specific forms of cyclin E. Results: The PR HR+/HER2- and TN breast cancer cell lines, unresponsive to palbociclib, exhibited significantly increased sensitivity to BLU-222. The combination of BLU-222 and palbociclib in all four cell lines revealed a robust synergistic effect in PR cells, inducing enhanced apoptosis and cell cycle alterations in G1 or G2/M phases. Treatment with BLU-222 and palbociclib demonstrated substantial antitumor activity in all six PDX models and the cyclin E high TN transgenic models, surpassing the effects of individual treatments in each model. This combination led to lasting tumor regression and extended survival, even after treatment discontinuation. Mechanistically, treatment with BLU-222, alone or combined with palbociclib, induced the expression of p21 and/or p27 in all in vivo models, which we hypothesize sensitizes tumors to palbociclib. In vitro, the CRISPR knockout of p21 or p27 in MCF-7 PR cells abolished the synergistic activity of BLU-222 and palbociclib, confirming the crucial involvement of p21 and p27 in influencing the treatment's effectiveness. Conclusions: We found robust activity of the CDK2 inhibitor, BLU-222 when combined with CDK4/6is in resistant HR+/HER2- and TN breast cancer cell lines and in vivo models. These results support the potential clinical utility of BLU-222 in combination with CDK4/6is for the treatment of both subtypes of breast cancer. Citation Format: Linjie Luo, Yan Wang, Tuyen Bui, Mi Li, Serena Kim, Juliana Navarro-Yepes, Nicole M. Kettner, Debasish Tripathy, Kelly K. Hunt, Kerrie Faia, Khandan Keyomarsi. Anti-tumor activity of CDK2 inhibitor BLU-222 in combination with CDK4/6 inhibitors for overcoming resistance in HR positive and triple negative metastatic breast cancers models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4622.

Abstract 3707: Characterization of estrogen receptor expression on CTCs during CDK4/6i treatment in HR+/HER2- metastatic breast cancer: Results from the PACE phase II study

Abstract Background Circulating tumor cells (CTCs) are associated with worse survival in metastatic breast cancer (MBC). The PACE study (NCT03147287) enrolled patients (pts) with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) MBC after progression on CDK4/6 inhibitor (CDK4/6i) and endocrine therapy, and randomized pts to fulvestrant (ful) alone; ful with palbociclib; or ful, palbociclib, and avelumab [Mayer et al., SABCS 2022]. Pts with ≥5 CTCs appear to derive benefit from treatment combinations vs ful alone. In this feasibility analysis, we evaluated estrogen receptor (ER) expression on CTCs to further investigate their monitoring/predictive potential.Methods Blood samples were collected from pts enrolled in the PACE study at: baseline (T1), first tumor assessment (T2) and progression (T3). CTCs were enumerated using the CellSearch platform and samples were classified as CTChigh or CTClow (cutoff ≥5 CTCs). CTChigh samples underwent ER expression quantification with the ACCEPT software [Zeune et al. 2017]. ER expression intensity (ERint) was measured through the Marker Characterization function for each CTC; the average was calculated for each sample. Results From the 220 enrolled pts, a total of 409 samples (166, 153 and 90 collected at T1, T2, and T3, respectively) were analyzed for ER. Overall, 236 samples were CTClow (T1 = 91, T2 = 97, T3 = 48) and 173 were CTChigh (T1 =75, T2 =56, T3 =42). ER was quantified on 3928, 1853, and 2209 CTCs collected at T1, T2, and T3, respectively. Overall, 32.2% of CTCs were ER negative (ER-).The samples’ median ERint showed a numerical decrease from T1 to T3 (T1 = 7.27, T2 = 4.58, T3 = 1.31). Similarly, the % of ER positive (ER+) CTCs per sample decreased from baseline to progression: 26.3%, 14.0%, 5.5% at T1, T2, T3, respectively.CTChigh samples were then classified as ER+ or ER- (ER+ = mean ER_int >0). At T1, 91 (54.8%) samples were CTClow, 67 (40.4%) were CTChigh/ER+, and 8 (4.8%) were CTChigh/ER-. At progression, 48 (53.3%) samples were CTClow, 26 (28.9%) were CTChigh/ER+, and 16 (17.8%) samples were CTChigh/ER-. A numerical increase in the proportion of CTChigh/ER- samples was thus observed from baseline to progression (4.8 to 17.8%).Conclusions Herein we demonstrated the feasibility of evaluating and quantify ER expression on CTCs with a software-assisted approach to reduce inter-operator variability. In the PACE study, ER expression on CTCs was decreased at progression compared to baseline, possibly suggesting a mechanism associated with treatment resistance. Future analyses are needed to investigate the correlation between ER expression levels on CTCs with survival and treatment response. This, together with information about the mutational status of ESR1 by ctDNA sequencing, might provide a new perspective on the development of resistance to ful and CDK4/6i in HR+/HER2- MBC. Citation Format: Mara Serena Serafini, Carolina Reduzzi, Lorenzo Gerratana, Rinath Jeselsohn, Reshma L. Mahtani, Cynthia X. Ma, Angela DeMichele, Jane Lowe Meisel, Kathy D. Miller, Yara Abdou, Elizabeth C. Riley, Rubina Qamar, Priyanka Sharma, Sonya Reid, Harold J. Burstein, Sara M. Tolaney, Michelle K. DeMeo, Yuan Liu, Eric Gauthier, Yue Ren, Meredith M. Regan, Huiping Liu, Erica L. Mayer, Massimo Cristofanilli. Characterization of estrogen receptor expression on CTCs during CDK4/6i treatment in HR+/HER2- metastatic breast cancer: Results from the PACE phase II study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3707.

Abstract 2429: Longitudinal ctDNA monitoring using a high sensitivity tumor-informed assay in patients with metastatic HR+/HER2− breast cancer receiving endocrine therapy and CDK4/6 inhibitors

Abstract BACKGROUND: ctDNA burden is prognostic and its dynamics during therapy may permit innovative strategies in metastatic breast cancer (mBC). Previous analyses have focused on driver genes, with limited sensitivity to quantify depth of response or detect early molecular progression. To enable sensitive, quantitative ctDNA monitoring, we analyzed samples from a cohort of HR+/HER2− mBC pts treated with endocrine therapy (ET) and CDK4/6 inhibitors (CDKi) using RaDaR, a patient-specific assay that tracks tumor-informed truncal variants independent of driver status with a limit of detection of 0.001%. METHODS: HR+/HER2− mBC pts receiving standard ET + CDKi were enrolled in a prospective observational cohort from 2018. Plasma samples were collected at baseline (BL), within 30 days (d), and ~q3 months (mo) with radiological scans. RaDaR was performed on all samples for pts with tissue available for WES (as per assay). Clinico-pathological variables were collected. Endpoints were time to treatment failure (TTF) and overall survival (OS). RESULTS: 51 pts were available for analysis. Median age was 60 years (range 38-88), 63% had visceral disease, and most pts were treated as first (75%) or second (20%) line. RaDaR was successful on 43 pts, with the rest dropping out due to failed WES (1) or panel failure (7). Archival tissue WES was used to design bespoke assays targeting a median 48 (23-52) variants in 248 samples, with a median of 5 samples/pt (2-14) collected over a median follow up of 27.8 (0.9-64.3) mo. ctDNA was detected in 91% (39/43) of BL and 70% (174/248) of all samples, with a median estimated variant allele fraction (eVAF) of 0.05%. Higher BL eVAFs were associated with liver disease (p=0.02) and with shorter TTF (p<0.01 adjusted by age and visceral disease) and OS (p<0.01). 36/39 pts had ≥1 sample with ctDNA decrease from BL, occurring within the first mo of therapy in 77% (24/31) of pts with available early samples. In 13 pts ctDNA became undetectable (‘suppressed’) at a median time (T) of 157 d (14-343). Among 25 pts with treatment failure (TF), the median T from the last negative/decreasing eVAF to TF was 163 d (0-943). Only 3 pts with TF had prior negative ctDNA, occurring with a median lead T of 198 d (119-595). Among ongoing pts, the median T from the last ctDNA decrease to last follow up was 317 d (163-1508), and from the last negative ctDNA sample was 352 d (163-993). CONCLUSION: RaDaR enabled sensitive ctDNA monitoring in 91% HR+/HER2− mBC pts on ET + CDK4/6i. ctDNA levels were prognostic and fell rapidly with therapy, but suppression occurred in less than one-third of pts, taking a median of 5.2 mo. Pts with negative/decreasing ctDNA may require less frequent clinical/radiographic tumor evaluations. Highly sensitive ctDNA tracking may inform prognostic assessments, follow up strategies, and innovative interventional trial designs. Citation Format: Jesus Fuentes-Antras, Mitchell J. Elliott, Philippe Echelard, Aaron Dou, Zachary Veitch, Philippe L. Bedard, Eitan Amir, Michelle B. Nadler, Nicholas Meti, Nancy Gregorio, Elizabeth Shah, Emily Van de Laar, Celeste Yu, Lisa Gates, Clodagh Murray, Christopher G. Smith, Amber Chevalier, Lillian L. Siu, Hal K. Berman, David W. Cescon. Longitudinal ctDNA monitoring using a high sensitivity tumor-informed assay in patients with metastatic HR+/HER2− breast cancer receiving endocrine therapy and CDK4/6 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2429.

Abstract 7630: Therapeutic relevance of systemic immune status to treatment effects of abemaciclib

Abstract Background: The addition of a CDK4/6 inhibitor (CDK4/6i) on endocrine therapies (ET) has been currently a standard treatment for hormone receptor (HR)-positive HER2-negative metastatic breast cancers (MBCs). Especially abemaciclib has been implicated in multiple mechanisms of action including cell cycle arrest, senescence. In addition, immunomodulatory effects have been suggested but are not yet clear. The aim of the study was to investigate the clinical relevance of abemaciclib to systemic immune status by examining the status of peripheral immune cells in patients with MBC who received abemaciclib plus ET. Methods: Patients with MBC who were treated with abemaciclib plus ET in our hospital were included in this study. Response was evaluated by RECIST 1.1. Peripheral blood at the initiation of abemaciclib treatment, 4 weeks, 12 weeks and 24weeks after abemaciclib treatment initiation were analyzed using mass cytometry by time of flight (CyTOF). Changes of cellular densities of each immune cell and survival by immune cell density were analyzed without calculating p values because of small sample number. Results: Twenty patients were enrolled, among whom 14 and 6 received an aromatase inhibitor and fulvestrant, respectively, as concomitant ET. 7 premenopausal patients received an LH-RH analogue. The analysis of serial blood collection showed an increase in CD8-positive T cell density by 12.5% and conversely a decrease in monocyte density by 52.3% at 4 weeks after treatment initiation. In responders (N = 10), an increase of the ratio between CD8-positive T cell density and Treg density (CD8/Treg) at 4 weeks after the treatment initiation were observed (65.2%), while not in non-responders (7.3%). In the analysis of the blood samples at the treatment initiation, lower Treg density and lower NK density at treatment initiation indicated longer PFS than the counterparts (median PFS for Treg: low, not reached; high, 8.8 months; for NK: low, not reached; high, 8.8 months). Conclusion: An increase of CD8/Treg at 4 weeks after the treatment initiation was associated with a good response in patients with HR-positive HER2-negative MBC treated with abemaciclib plus ET. Our results suggest an association between systemic immune status and clinical outcome in MBC patients who received abemaciclib plus ET treatment. A larger study is needed to validate the results. Citation Format: Takayuki Ueno, Shigehisa Kitano, Takayuki Kobayashi, Makiko Yamashita, Fumikata Hara, Yukinori Ozaki, Meiko Nishimura, Toshimi Takano, Naoko Toriguchi, Tsutomu Kawaguchi. Therapeutic relevance of systemic immune status to treatment effects of abemaciclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7630.

PACE: A Randomized Phase II Study of Fulvestrant, Palbociclib, and Avelumab After Progression on Cyclin-Dependent Kinase 4/6 Inhibitor and Aromatase Inhibitor for Hormone Receptor-Positive/Human Epidermal Growth Factor Receptor-Negative Metastatic Breast Cancer.

Cyclin-dependent kinase (CDK) 4/6 inhibitors (CDK4/6is) are an important component of treatment for hormone receptor-positive/human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (MBC), but it is not known if patients might derive benefit from continuation of CDK4/6i with endocrine therapy beyond initial tumor progression or if the addition of checkpoint inhibitor therapy has value in this setting. The randomized multicenter phase II PACE trial enrolled patients with hormone receptor-positive/HER2- MBC whose disease had progressed on previous CDK4/6i and aromatase inhibitor (AI) therapy. Patients were randomly assigned 1:2:1 to receive fulvestrant (F), fulvestrant plus palbociclib (F + P), or fulvestrant plus palbociclib and avelumab (F + P + A). The primary end point was investigator-assessed progression-free survival (PFS) in patients treated with F versus F + P. Overall, 220 patients were randomly assigned between September 2017 and February 2022. The median age was 57 years (range, 25-83 years). Most patients were postmenopausal (80.9%), and 40% were originally diagnosed with de novo MBC. Palbociclib was the most common previous CDK4/6i (90.9%). The median PFS was 4.8 months on F and 4.6 months on F + P (hazard ratio [HR], 1.11 [90% CI, 0.79 to 1.55]; P = .62). The median PFS on F + P + A was 8.1 months (HR v F, 0.75 [90% CI, 0.50 to 1.12]; P = .23). The difference in PFS with F + P and F + P + A versus F was greater among patients with baseline ESR1 and PIK3CA alterations. The addition of palbociclib to fulvestrant did not improve PFS versus fulvestrant alone among patients with hormone receptor-positive/HER2- MBC whose disease had progressed on a previous CDK4/6i plus AI. The increased PFS seen with the addition of avelumab warrants further investigation in this patient population.

Exemestane plus everolimus and palbociclib in metastatic breast cancer: clinical response and genomic/transcriptomic determinants of resistance in a phase I/II trial

The landscape of cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) resistance is still being elucidated and the optimal subsequent therapy to overcome resistance remains uncertain. Here we present the final results of a phase Ib/IIa, open-label trial (NCT02871791) of exemestane plus everolimus and palbociclib for CDK4/6i-resistant metastatic breast cancer. The primary objective of phase Ib was to evaluate safety and tolerability and determine the maximum tolerated dose/recommended phase II dose (100 mg palbociclib, 5 mg everolimus, 25 mg exemestane). The primary objective of phase IIa was to determine the clinical benefit rate (18.8%, n = 6/32), which did not meet the predefined endpoint (65%). Secondary objectives included pharmacokinetic profiling (phase Ib), objective response rate, disease control rate, duration of response, and progression free survival (phase IIa), and correlative multi-omics analysis to investigate biomarkers of resistance to CDK4/6i. All participants were female. Multi-omics data from the phase IIa patients (n = 24 tumor/17 blood biopsy exomes; n = 27 tumor transcriptomes) showed potential mechanisms of resistance (convergent evolution of HER2 activation, BRAFV600E), identified joint genomic/transcriptomic resistance features (ESR1 mutations, high estrogen receptor pathway activity, and a Luminal A/B subtype; ERBB2/BRAF mutations, high RTK/MAPK pathway activity, and a HER2-E subtype), and provided hypothesis-generating results suggesting that mTOR pathway activation correlates with response to the trial’s therapy. Our results illustrate how genome and transcriptome sequencing may help better identify patients likely to respond to CDK4/6i therapies.

PRMT5 is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.

Patient-derived tumor xenograft study with CDK4/6 inhibitor plus AKT inhibitor for the management of metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive malignancy with poor outcomes. To investigate novel therapeutic strategies, we characterized three new metastatic prostate cancer PDTX models and developed 3D spheroids from each to investigate molecular targeted therapy combinations including CDK4/6 inhibitors (CDK4/6i) with AKT inhibitors (ATKi). Metastatic prostate cancer tissue was collected and three PDTX models were established and characterized using WES. PDTX 3-D spheroids were developed from these three PDTXs to show resistance patterns and test novel molecular targeted therapies. CDK4/6i's were combined with AKTi's to assess synergistic antitumor response to prove our hypothesis that blockade of AKT overcomes drug resistance to CDK4/6 inhibitor. This combination was evaluated in PDTX 3-D spheroids and in vivo experiments with responses measured by tumor volumes, PSA and Ga-68 PSMA-11 PET-CT imaging. We demonstrated CDK4/6i's with AKTi's possess synergistic antitumor activity in three mCRPC PDTX models. These models have multiple unique pathogenic and deleterious genomic alterations with resistance to single agent CDK4/6i's. Despite this, combination therapy with AKTi's was able to overcome resistance mechanisms. The IHC and Western blot analysis confirmed on target effects, while tumor volume, serum PSA ELISA, and radionuclide imaging demonstrated response to therapy with statistically significant SUV differences seen with Ga-68 PSMA-11 PET-CT. These preclinical data demonstrating antitumor synergy by overcoming single agent CDK 4/6i as well as AKTi drug resistance provide the rational for a clinical trial combining a CDK4/6i with an AKTi in mCRPC patients whose tumor expresses wild type RB1.

MYC induces CDK4/6 inhibitors resistance by promoting pRB1 degradation

CDK4/6 inhibitors (CDK4/6i) show anticancer activity in certain human malignancies, such as breast cancer. However, their application to other tumor types and intrinsic resistance mechanisms are still unclear. Here, we demonstrate that MYC amplification confers resistance to CDK4/6i in bladder, prostate and breast cancer cells. Mechanistically, MYC binds to the promoter of the E3 ubiquitin ligase KLHL42 and enhances its transcription, leading to RB1 deficiency by inducing both phosphorylated and total pRB1 ubiquitination and degradation. We identify a compound that degrades MYC, A80.2HCl, which induces MYC degradation at nanomolar concentrations, restores pRB1 protein levels and re-establish sensitivity of MYC high-expressing cancer cells to CDK4/6i. The combination of CDK4/6i and A80.2HCl result in marked regression in tumor growth in vivo. Altogether, these results reveal the molecular mechanisms underlying MYC-induced resistance to CDK4/6i and suggest the utilization of the MYC degrading molecule A80.2HCl to potentiate the therapeutic efficacy of CDK4/6i.

Liquid biopsy utilizing miRNA in patients with advanced breast cancer treated with cyclin‑dependent kinase 4/6 inhibitors.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) are the mainstay of treatment of hormone receptor+/human epidermal growth factor receptor 2-patients with advanced breast cancer (ABC). Despite improvements in overall survival, most patients experience disease progression. Biomarkers derived from a liquid biopsy are appealing for their potential to detect resistance to treatment earlier than computed tomography imaging. However, clinical data concerning microRNAs (miRNAs/miRs) in the context of CDK4/6is are lacking. Thus, the present study assessed the use of miRNAs in patients with ABC treated with CDK4/6is. Patients treated for ABC with CDK4/6is between June and August 2022 were eligible. miRNA expression analyses were performed using a TaqMan™ low-density miRNA array. A total of 80 consecutive patients with ABC treated with CDK4/6is at Maria Sklodowska-Curie National Research Institute of Oncology (Gliwice, Poland) were assessed, with 14 patients diagnosed with progressive disease at the time of sampling, 55 patients exhibited clinical benefit from CDK4/6i treatment and 11 patients were at the beginning of CDK4/6i treatment. Patients with disease progression had significantly higher levels of miR-21 (P=0.027), miR-34a (P=0.011), miR-193b (P=0.032), miR-200a (P=0.027) and miR-200b (P=0.003) compared with patients who benefitted from CDK4/6i treatment. Significantly higher levels of miR-34a expression were observed in patients with progressive disease than in patients beginning treatment (P=0.031). The present study demonstrated the potential innovative role of circulating miRNAs during CDK4/6i treatment. Plasma-based expression of miR-21, -34a, -193b, -200a and -200b effectively distinguished patients with ABC who responded to CDK4/6i treatment from patients who were resistant. However, longitudinal studies are required to verify the predictive and prognostic potential of miRNA.

CDK4/6i-treated HR+/HER2- breast cancer tumors show higher ESR1 mutation prevalence and more altered genomic landscape

As CDK4/6 inhibitor (CDK4/6i) approval changed treatment strategies for patients with hormone receptor-positive HER2-negative (HR+/HER2-) breast cancer (BC), understanding how exposure to CDK4/6i affects the tumor genomic landscape is critical for precision oncology. Using real-world data (RWD) with tumor genomic profiling from 5910 patients with metastatic HR+/HER2- BC, we investigated the evolution of alteration prevalence in commonly mutated genes across patient journeys. We found that ESR1 is more often altered in tumors exposed to at least 1 year of adjuvant endocrine therapy, contrasting with TP53 alterations. We observed a similar trend after first-line treatments in the advanced setting, but strikingly exposure to aromatase inhibitors (AI) combined with CDK4/6i led to significantly higher ESR1 alteration prevalence compared to AI alone, independent of treatment duration. Further, CDK4/6i exposure was associated with higher occurrence of concomitant alterations in multiple oncogenic pathways. Differences based on CDK4/6i exposure were confirmed in samples collected after 2L and validated in samples from the acelERA BC clinical trial. In conclusion, our work uncovers opportunities for further treatment personalization and stresses the need for effective combination treatments to address the altered tumor genomic landscape following AI+CDK4/6i exposure. Further, we demonstrated the potential of RWD for refining patient treatment strategy and guiding clinical trial design.

Cracking the Genomic Code of CDK4/6 Inhibitor Resistance.

The therapeutic approach to metastatic hormone-receptor positive, human epidermal growth factor-2 negative metastatic breast cancer (HR+/HER2- MBC) has evolved rapidly over recent years. The cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have become first-line targeted agents of choice, in combination with an anti-estrogen. Simultaneously, the clinical landscape of therapeutic options has been rapidly shifting, with novel anti-estrogens, signal transduction inhibitors, and next-generation CDK inhibitors in various staging of development. Given these dynamic changes, understanding the genomic and molecular landscape of resistance to currently available anti-estrogen therapy and CDK4/6 inhibitors represents a major focus of translational breast cancer research globally.

Comparative overall survival of CDK4/6 inhibitors in combination with endocrine therapy in advanced breast cancer

Individual trials of abemaciclib, palbociclib, and ribociclib show a similar impact on progression-free survival yet differing statistical significance for overall survival (OS). A robust comparative evaluation of OS, safety, and tolerability of the three drugs is warranted. A systematic literature search identified phase 3 randomized clinical trials reporting OS of CDK4/6 inhibitors (CDK4/6i) in combination with endocrine therapy in ER-positive/HER2-negative advanced breast cancer. Trial-level data on OS and common and serious adverse events (AE) were extracted for each drug. In the absence of direct comparisons, a network meta-analysis was performed to evaluate pairwise comparative efficacy, safety, and tolerability of each of the CDK4/6i. Seven studies comprising of 4415 patients met the inclusion criteria. Median follow-up was 73.3 months (range: 48.7–97.2 months). There were no statistically significant differences in OS between any of the CDK4/6i. Compared to palbociclib, ribociclib and abemaciclib both showed significantly higher GI toxicity (grade 1–2 vomiting OR 1.87 [95% CI 1.37–2.56] and OR 2.27 [95% CI 1.59–3.23] respectively). Compared to palbociclib, abemaciclib was associated with more grade 3–4 diarrhea OR 118.06 [95% CI 7.28–1915.32]. In contrast, palbociclib was associated with significantly more neutropenia than ribociclib and abemaciclib but significantly lower risk of grade 3–4 infections. Abemaciclib had significantly less grade 3–4 transaminitis and grade 3–4 neutropenia than ribociclib. Treatment discontinuation and death due to AE were significantly higher with abemaciclib than palbociclib and ribociclib. There is no statistically significant difference in OS between CDK4/6i despite differing statistical significance levels of individual trials. Real-world data analyses may help to identify if there is a meaningful inter-drug difference in efficacy. Significant differences between CDK4/6i are observed for safety and tolerability outcomes.