CDK4 Pathway Research Articles

Therapeutic Prospects of Abemaciclib for Patients with Endometrial Cancer.

Endometrial cancer (EC) is a common gynecologic malignancy with a rising incidence due to obesity, comorbid conditions, and related lifestyle factors. The standard of care for primary disease consists of surgical resection with/without chemotherapy ± radiotherapy for select patients. Recurrence is common in patients with advanced-stage disease and/or high-risk features, who primarily are treated with systemic therapy. The identification of novel targets in malignant EC has led to the development of wide-range inhibitors. Abemaciclib is an orally active unique cyclin-dependent kinase (CDK) inhibitor, selective for the CDK4 and CDK6 cell cycle pathways. This agent has potential anti-neoplastic activity and is indicated in combination with various therapies such as endocrine therapy, aromatase inhibitors, and hormone therapies, primarily in breast cancer (BC). Herein, we sought to summarize the biochemical/pharmacological properties of abemaciclib and its therapeutic potential in EC. While the therapeutic role(s) of abemaciclib was fairly established in a subset of patients with advanced/metastatic BC through the pivotal MONARCH trials, its attributes and clinical utility in EC are limited. Thus, based on some promising pre-clinical/translational insights and a recent phase II study, we highlight abemaciclib's properties and potential clinical usefulness in patients with EC, particularly in recurrent estrogen-receptor-positive cases.

Exploration and biological evaluation of 20-vinyl pregnenes: A step forward toward selective modulators of the estrogen receptor α signaling for breast cancer treatment.

A series of D-ring modified steroids bearing a vinyl ketone pendant were synthesized and evaluated for antiproliferative activity against breast cancer cell line and cytochromes P450. The lead compound, 21-vinyl 20-keto-pregnene (2f) (IC50 = 2.4 µM), was shown to be a promising candidate for future anticancer drug design, particularly against estrogen receptor α (ERα)-positive breast cancer. The lead compound was found to have a significant effect on the signaling pathways in parental and 4-hydroxytamoxifen-resistant cells. Compound 2f modulated the ERK, cyclin D1, and CDK4 pathways and blocked the expression of ERα, the main driver of breast cancer growth. Compound 2f significantly reduced 17β-estradiol-induced progesterone receptor expression. Accumulation of cleaved poly(ADP-ribose) polymerase in cells treated with compound 2f indicated induction of apoptosis. The selectivity analysis showed that lead compound 2f produces no significant effects on cytochromes P450, CYP19A1, CYP21A2, and CYP7B1.

Design and synthesis of phosphoryl-substituted steroidal pyridazines (Pho-STPYRs) as potent estrogen receptor alpha inhibitors: targeted treatment of hormone-dependent breast cancer cells.

Estrogen receptor alpha (ERα) is an important target for the discovery of new therapeutic drugs against hormone-dependent breast cancer. A series of phosphoryl-substituted steroidal pyridazines (Pho-STPYRs) were synthesized and biologically evaluated as potent ERα inhibitors. Pho-STPYRs showed cytotoxicity against breast cancer cells with IC50 values of 5.9 μM and higher. Pho-STPYRs 33 and 34 [IC50 (MCF7) = 6.5 and 5.9 μM, respectively] were found to block the expression of ERα, the main driver of breast cancer growth, and modulate the ERK, cyclin D1, and CDK4 pathways. Compound 34 showed selectivity, anti-estrogenic potency and high antiproliferative efficacy in combination with the AKT inhibitor. Molecular docking was used to more accurately define the binding mode of lead compounds 33 and 34 to ERα. The selectivity analysis showed that lead compounds 33 and 34 produce no effects on cytochromes P450, including CYP7A1, CYP7B1, CYP17A1, CYP19A1, and CYP21A2. In a word, Pho-STPYRs 33 and 34 are promising ERα inhibitors for the treatment of hormone-dependent breast cancer.

Mycotoxins and cellular senescence: the impact of oxidative stress, hypoxia, and immunosuppression.

Mycotoxins induce oxidative stress, hypoxia, and cause immunosuppressive effects. Moreover, emerging evidence show that mycotoxins have a potential of inducing cellular senescence, which are involved in their immunomodulatory effects. Mycotoxins upregulate the expression of senescence markers γ-H2AX, senescence-associated β-galactosidase, p53, p16, and senescence-associated secretory phenotype (SASP) inflammatory factors. Moreover, mycotoxins cause senescence-associated cell cycle arrest by diminishing cyclin D1 and Cdk4 pathways, as well as increasing the expression of p53, p21, and CDK6. Mycotoxins may induce cellular senescence by activating reactive oxygen species (ROS)-induced oxidative stress. In addition, hypoxia acts as a double-edged sword on cell senescence; it could both act as the stress-induced senescence and also hinder the onset of cellular senescence. The SASP inflammatory factors have the ability to induce an immunosuppressive environment, while mycotoxins directly cause immunosuppression. Therefore, there is a potential relationship between mycotoxins and cellular senescence that synergistically cause immunosuppression. However, most of the current studies have involved the effect of mycotoxins on cell cycle arrest, but only limited in-depth research has been carried out to link the occurrence of this condition (cell cycle arrest) with cellular senescence.

Harmine and 7,8-dihydroxyflavone synergistically suitable for amyotrophic lateral sclerosis management: An in silico study

Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by progressive degeneration of both upper and lower motor neurons, resulting in paralysis and eventually leads to death from respiratory failure typically within 3 to 5 years of symptom onset. The aim of this work was to predict the pharmacokinetics and identify unique protein targets that are associated with potential anti-ALS phytochemicals and FDA-approved drugs, by in silico approaches.
 Materials and methods: Standard computational tools (webserver and software) were used, and the methods used are clustering analysis, pharmacokinetics and molecular target predictions, and molecular docking simulation.
 Results and discussion: The results show that riluzole, β-asarone, cryptotanshinone, harmine and 7,8-dihydroxyflavone have similar pharmacokinetics properties. Riluzole and harmine show 95% probability of target on norepinephrine transporter. Huperzine-A and cryptotanshinone show 100% probability of target on acetylcholinesterase. 7,8-dihydroxyflavone shows 35% probability of target on several carbonic anhydrases, 40% probability of target on CYP19A1, and 100% probability of target on inhibitor of nuclear factor kappa B kinase beta subunit and neurotrophic tyrosine kinase receptor type 2, respectively. Harmine also shows 95% probability of target on dual specificity tyrosine-phosphorylation-regulated kinases, threonine-protein kinases (haspin and PIM3), adrenergic receptors, cyclin-dependent kinases (CDK5 and CDK9), monoamine oxidase A, casein kinase I delta, serotonin receptors, dual specificity protein kinases (CLK1, CLK2, and CLK4), and nischarin, respectively. Also, the results of gene expression network show possible involvement of CDK1, CDK2, CDK4, ERK1, ERK2 and MAPK14 signaling pathways. This study shows that riluzole and harmine have closely similar physicochemical and pharmacokinetics properties as well as molecular targets, such as norepinephrine transporter (SLC6A2). Harmine, huperzine-A and cryptotanshinone could modulate acetylcholinesterase (AChE), which is involved in ALS-pathogenesis. The impact of 7,8-dihydroxyflavone on several carbonic anhydrases (CA) I, II, VII, IX, XII, and XIV, as well as CYP19A1, could help in remediating the respiratory failure associated with ALS.
 Conclusion: Overall, harmine is found to be superior to riluzole, and the combination of harmine with 7,8-dihydroxyflavone can provide more effective treatment for ALS than the current regime. Further work is needed to validate the predicted therapeutic targets of harmine identified in this study on ALS model or clinical trials, using in silico, in vitro and in vivo techniques.
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Endometrial Stromal Sarcomas With BCOR Internal Tandem Duplication and Variant BCOR/BCORL1 Rearrangements Resemble High-grade Endometrial Stromal Sarcomas With Recurrent CDK4 Pathway Alterations and MDM2 Amplifications.

The distinction between low-grade and high-grade endometrial stromal sarcomas (LGESS, HGESS) is increasingly defined by genetics. Recently, variant genomic alterations involving BCOR or BCORL1 have been reported in endometrial stromal sarcoma (ESS), although it remains unclear whether these justify a diagnosis of LGESS or HGESS. In this study, we describe clinicopathologic and molecular features of ESS with such alterations to help clarify their classification in the spectrum of ESS. We collected a cohort of 13 ESS harboring variant alteration involving BCOR (6 with internal tandem duplication, 1 with EP300::BCOR fusion, 1 with BCOR::LPP fusion) and BCORL1 ( 4 with JAZF1::BCORL1 fusion, 1 with EPC1::BCORL1 fusion). The median patient age at primary diagnosis was 51 years (range: 18 to 70 y). Median tumor size at primary diagnosis was 9.3 cm (range: 4.5 to 21 cm), and extrauterine disease spread (stage IIIB-C) was present in 27%. The tumors were composed of round to spindled cells with cellularity and cytologic atypia ranging from mild to marked and a median mitotic count of 18/10 HPFs (range: 2 to 85/10 HPFs). At least focally myopermeative growth was noted in 8/8 assessable cases. Of 12 patients with follow-up data (median: 25 mo), 4 patients died of disease and 3 were alive with recurrent disease. Unsupervised hierarchical clustering of DNA methylation data together with a large cohort of uterine mesenchymal tumors that included YWHAE::NUTM2 and Z C3H7B::BCOR HGESS and molecularly confirmed LGESS revealed a common methylation signature for all ESS with variant BCOR and BCORL1 alterations and HGESS with YWHAE::NUTM2 and ZC3H7B::BCOR gene fusion. Copy number analysis revealed amplifications of CDK4 and MDM2 , as well as homozygous deletions of CDKN2A/B and NF1 in a subset of tumors. Our results indicate that ESS with BCOR internal tandem duplication and variant BCOR and BCORL1 rearrangements clinically and molecularly resemble conventional HGESS.

Regulation Effect of miR-34a Expression on Radiosensitivity of Lung Adenocarcinoma Cells by Targeting Bcl-2 and CDK4/6 Signaling Pathways

Objective: Radiotherapy has been widely used to treat lung cancer. However, non-small lung cancer cells are insensitive to radiation, diminishing their radiotherapy effects. Although the radiosensitivity of the non-small lung cancer cells was reported to be enhanced through regulating miR-34a, the regulation effects of miR-34a expression on radiosensitivity of lung adenocarcinoma cells through target genes CDK4, CDK6, CyclinD1, and Bcl-2/Bax have not been systematically investigated. Methods: In this study, we investigated the effect of miR-34a expression on the Bcl-2, CDK4, and CDK6 pathways in lung adenocarcinoma cells, to provide new insights into the sensitization treatment of lung cancer. We first studied the effect of miR-34a expression on H1299 and A549 cell activity. Then to investigate the mechanisms of radiosensitivity, we focused on apoptosis, cell cycle, and target genes. Results: We find that overexpression of miR-34a in lung adenocarcinoma cells inhibits cell activity, and improves radiosensitivity. Specifically, overexpression of miR-34a suppresses the expression of target genes CDK4, CDK6, CyclinD1, and Bcl-2/Bax, which leads to cell cycle arrest and promotes apoptosis of lung adenocarcinoma cells. Conclusions: Overall, our results demonstrate that the overexpression of miR-34a enhances the radiosensitivity of lung adenocarcinoma cells, indicating that miR-34a is a sensitizer for lung adenocarcinoma radiotherapy.

Abstract 377: Molecular features-based model for predicting benefit from Bevacizumab combined with first-generation EGFR-tyrosine kinase inhibitor

Abstract Introduction: Two phase 3 clinical studies, NEJ026 and CTONG1509, have consistently demonstrated better anti-tumor activity of adding bevacizumab to erlotinib regimen in the treatment of patients with epidermal growth factor receptor (EGFR)-mutant advanced non-squamous non-small-cell lung cancer (NSCLC); however, the subset of patients that benefits more with the addition of bevacizumab to their EGFR-TKI regimen still remains to be identified. In this study, we aimed to investigate the clinical and molecular factors that affect the efficacy of first-generation EGFR-TKI regimen with or without bevacizumab as first-line treatment of patients with EGFR-mutant NSCLC. Methods: A total of 176 patients with EGFR-mutant stage IIIB-IV relapsed or metastatic NSCLC and received first-generation EGFR-TKI gefitinib or erlotinib monotherapy (T; n=88) or combined with bevacizumab (A+T; n=88) were included in our study. The two groups had comparable baseline clinical characteristics. Retrospective analysis was performed on clinical data, survival outcomes, and mutation profiles at baseline and progression. A nomogram model to predict PFS was constructed with the following variables identified from Cox multivariate analysis: treatment group (A+T) and mutations in EGFR L858R, exon 19 deletion, TP53, CDK4, FGF3, and NFkB signaling pathway. Another nomogram was constructed to predict overall survival with treatment group (A+T) and mutations in TP53 signaling pathway as the variables. Results: As compared to the T group, progression-free survival (PFS) was significantly longer among patients who received A+T and harbored concurrent EGFR amplification (16.1 vs 9.0 months; HR: 0.267, 95%CI: 0.110-0.651; p=0.004), and hotspot and loss-of-function mutations in TP53 (16.1 vs 8.0 months; HR: 0.215, 95%CI: 0.108-0.426; p<0.001). At progression, EGFR T790M, detected from 35% in the A+T group and 42% in the T group, was the major resistance mechanism in both groups. No statistical difference was found between the acquired resistance mechanisms of the two groups (p=0.787). Cox multivariate analysis demonstrated that receiving A+T is an independent predictor of longer PFS (HR: 0.51, 95%CI: 0.35-0.75; p=0.001) and overall survival (HR: 0.62, 95%CI: 0.38-0.99; p=0.045). Cross-validation of both nomograms using data from 144 evaluable patients in the cohort stratified into low-risk and high-risk groups based on the variables predicted the risk for disease progression (c-index: 0.676; p<0.001) and the risk for poor prognosis (c-index: 0.593; p<0.001) of the high-risk groups. Conclusions: Our study demonstrated the clinical value of the nomogram model in predicting the subset of patients who benefits from first-generation EGFR-TKI regimen with or without bevacizumab. Citation Format: Yongchang Zhang, Analyn Lizaso. Molecular features-based model for predicting benefit from Bevacizumab combined with first-generation EGFR-tyrosine kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 377.

Preliminary results of the abemaciclib arm in the Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT): A phase II platform trial using Bayesian adaptive randomization.

2014 Background: The cyclin D-CDK4/6-Rb pathway is activated in most glioblastomas. Abemaciclib is a potent CDK4/6 inhibitor with good brain penetration approved for HR+/HER2- breast cancer. In order to efficiently evaluate the potential impact of abemaciclib on overall survival (OS) in newly diagnosed glioblastoma and to simultaneously develop information regarding potential genomic biomarker associations, abemaciclib was included as an arm on the Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT) trial. INSIGhT is a phase II platform trial using response adaptive randomization and deep genomic profiling to more efficiently test experimental agents in MGMT unmethylated glioblastoma and potentially accelerate identification of novel therapies for phase III testing. Initial randomization was equal between abemaciclib, control, and two other experimental arms but subsequent randomization was adapted based on efficacy as determined by progression-free survival (PFS). Ineffective arms were discontinued and new arms added by protocol amendment. We report preliminary results for the abemaciclib arm which has completed accrual. Methods: Patients with newly diagnosed MGMT-unmethylated glioblastoma were randomized to receive either radiotherapy with concomitant and adjuvant temozolomide at standard doses or standard radiochemotherapy followed by adjuvant abemaciclib (150-200 mg orally BID). Treatment continued until progression or development of unacceptable toxicities. The primary endpoint was OS which was assessed using the log-rank test estimated via the Kaplan Meier method using a type I error of 5%. The hazard ratio (HR) was estimated using a cox proportional hazards model. Association between abemaciclib efficacy and cyclin D-CDK4/6-Rb pathway genomic alterations was also investigated. Results: There were 142 patients (69 control; 73 treated with abemaciclib). Abemaciclib was generally well-tolerated with no new toxicity signals identified. PFS was significantly longer (HR 0.67; p = 0.03, logrank test) with abemaciclib (median 6.54 months) compared to the control arm (median 5.88 months). For patients with activation of the CDK4 pathway the PFS HR was 0.64 (p-value = 0.04). However, there was no significant improvement in overall survival (HR 0.9; p-value > 0.05) between abemaciclib (median 15.5) compared to the control arm (median 15.5). Conclusions: Abemaciclib was well-tolerated and prolonged PFS but there is no evidence of an overall survival improvement compared to standard radiochemotherapy. Clinical trial information: NCT02977780.

Palbociclib in advanced acral melanoma with genetic aberrations in the cyclin-dependent kinase 4 pathway

BackgroundGenetic aberrations in the cyclin-dependent kinase (CDK)4 pathway occur in 82% of patients with acral melanoma (AM), which is the predominant subtype of melanoma in China. We aimed to evaluate the anti-tumour activity of palbociclib, a selective CDK4/6 inhibitor, in patients with advanced AM with CDK4 pathway gene aberrations. MethodsIn this phase II trial, patients with advanced AM with CDK4 or/and CCND1 gain or/and CDKN2A loss were treated with oral palbociclib (125 mg) on days 1–21 of a 28-day cycle. The primary end-point was overall response rate (ORR). Secondary end-points were progression-free survival (PFS), overall survival (OS), and treatment-related adverse events (TRAEs). Whole-exome sequencing and multiplex immunohistochemistry of the available formalin-fixed, paraffin-embedded samples of nine patients were analysed to explore the predictive biomarkers of palbociclib response. ResultsFifteen patients were enrolled. Three (20.0%) patients achieved tumour shrinkage at 8 weeks, including one with confirmed partial response. At data cut-off date, treatment was ongoing for one patient. The median PFS was 2.2 mo (range: 1.5–13.3 mo; 95% confidence interval [CI]: 1.9–2.5), and the median OS was 9.5 mo (range: 2.6–14.1 mo, 95% CI: 5.7–13.4). Eight patients died due to disease progression. The most common TRAEs were leukopenia (87%; Grade III/IV, 27%), neutropenia (80%; grade III/IV, 27%), and fatigue (53%; grade III/IV, 7%). Significant JAK2 deletions and SH2B3 amplifications were observed in patients who did not achieve any clinical benefit (CB) with palbociclib treatment. MCM7 amplification or protein expression level was found to be associated with CB. ConclusionsPalbociclib monotherapy demonstrated preliminary efficacy and an acceptable safety profile in advanced AM patients with CDK4 pathway aberrations. Patients with amplification or high protein levels of MCM7 were more prone to benefit from palbociclib. The JAK-STAT pathway might play a role in the mechanism of action of palbociclib in AM. Trial registration numberNCT03454919. The date of registrationMarch 6, 2018.

The tumor genetics of acral melanoma: What should a dermatologist know?

Dermatologists stand at the gateway of individualization of classification, treatment, and outcomes of acral melanoma patients. The acral melanoma genetic landscape differs in vital ways from that of other cutaneous melanomas. These differences have important implications in understanding pathogenesis, treatment, and prognosis. The selection of molecularly targeted therapy must be adapted for acral melanoma. It is also critical to recognize that tumor development is far more complex than an isolated event, reliably treated by a medication acting on a single target. Tumors exhibit intratumor genetic heterogeneity, metastasis may have different genetic or epigenetic features than primary tumors, and tumor resistance may develop because of the activation of alternative genetic pathways. Microenvironmental, immune, and epigenetic events contribute and sustain tumors in complex ways. Treatment strategies with multiple targets are required to effectively disrupt the tumor ecosystem. This review attempts to translate the current molecular understanding of acral melanoma into digestible concepts relevant to the practice of dermatology. The focus is tumor genetics defining potentially treatable cancer pathways, contextualized within the relevant pathologic and molecular features.

Remodelling of the bone marrow microenvironment by stromal hyaluronan modulates the malignancy of breast cancer cells

BackgroundHyaluronan (HA) is an abundant component of the bone marrow (BM) extracellular matrix. Here, we investigated the abnormal deposition of HA in the BM microenvironment and its remodelling in mediating the malignancy of breast cancer cells (BCCs).MethodsBCCs were transplanted into nude mice by intracardiac injection. The BCCs were cocultured with BM-derived stromal HS5 cells. Then, the abnormal metabolism of HA and its correlation with the malignant growth and the intracellular signalling pathways of the BCCs were investigated. After knockdown/out of the HA receptor CD44 in cancer cells by shRNA and CRISPR/Cas9, the mechanism was investigated in vivo through intratibial inoculation and in vitro by coculture with HS5 cells.ResultsThe malignancy of cancer cells was highly related to the degree of accumulation of HA in the BM. Further, stromal cell-derived HA, especially the mixed complex, significantly promoted the growth of BCCs and osteolysis by binding to the CD44 receptor. Additionally, the investigation of the underlying mechanism revealed that the PI3K, Cyclin D1, and CDK4 pathways were involved in the effect of bone stromal cell-derived HA on the BCC activities.ConclusionThese data suggested that HA in abnormal BM stroma might be a therapeutic candidate for bone metastasis of breast cancer.19kbyyRgNofaNy4kV-u_efVideo Graphical abstract

Genomic profiling of BCOR-rearranged uterine sarcomas reveals novel gene fusion partners, frequent CDK4 amplification and CDKN2A loss

ObjectiveGenomic alterations of BCOR via ZC3H7B-BCOR fusion or BCOR internal tandem duplication (ITD) define a subset of endometrial stromal sarcoma (ESS). The goals of this study were to: 1) determine the molecular landscape of BCOR-rearranged ESS, 2) to identify novel BCOR fusion gene partners in ESS and their associated clinicopathological characteristics, and 3) to potentially unravel targetable genomic alterations in BCOR-mutated ESS. MethodsA retrospective database search of a CLIA-certified molecular laboratory was performed for uterine sarcomas that contained BCOR rearrangements or BCOR ITD. The cases were previously assayed by comprehensive genomic profiling via both DNA- and RNA-based targeted next generation sequencing during the course of clinical care. Clinicopathological and genomic data was centrally re-reviewed. ResultsWe identify largest cohort of BCOR-rearranged ESS to date (n = 40), which included 31 cases with canonical ZC3H7B-BCOR fusion as well as 8 cases with novel BCOR gene rearrangement partners, such as BCOR-L3MBTL2, EP300-BCOR, BCOR-NUTM2G, BCOR-RALGPS1, BCOR-MAP7D2, RGAG1-BCOR, ING3-BCOR, BCOR-NUGGC, KMT2D-BCOR, CREBBP-BCOR and 1 case with BCOR internal rearrangement. Re-review of cases with novel rearrangements demonstrated sarcomas with spindle, epithelioid or small round cell components and frequent myxoid stromal change. Comprehensive genomic profiling revealed high frequency of CDK4 and MDM2 amplification in 38% and 45% of BCOR-rearranged cases, respectively, and homozygous deletion of CDKN2A, which encodes an inhibitor of CDK4 in 28% of cases. Notably, CDK4 and MDM2 amplification was absent in all cases from 15 different ESS cases harboring BCOR ITD. ConclusionsAlterations of CDK4 pathway members, for which targeted therapy is clinically available (i.e. palbociclib), via CDK4 amplification or CDKN2A loss, contributes to the pathogenesis of BCOR-rearranged uterine sarcomas, which may have therapeutic implications.

Abstract P4-05-10: Comparative analysis of genomic landscape reveals heterogeneity in HER2-positive primary breast cancers and residual disease following neoadjuvant therapy

Abstract Background: Neoadjuvant therapy is currently standard of care for patients with HER2-positive (HER2+) breast cancer. Patients with residual disease after neoadjuvant therapy have a higher risk of metastatic recurrence compared to patients achieving pathologic complete response (pCR). While both primary breast cancer and residual disease may harbor cancer cell subpopulations with high metastatic potential, residual disease after neoadjuvant targeted therapy may include larger subpopulations of therapy-resistant cells. A better understanding of the differences in genomic aberrations in residual disease following neoadjuvant therapy in comparison to primary breast cancer is essential to choice of novel post-neoadjuvant treatment strategy for HER2+ patients with residual disease. Methods: We are currently building a large ethnically diverse, breast cancer cohort with integrated genomic data. We analyzed clinical data of stage 1-3, HER2+ breast cancer. pCR after neoadjuvant therapy was defined as ypT0N0 or ypTisNo. Using the xT 595-gene panel (Tempus Labs, Inc.) with matched tumor-normal samples in a subset of the cohort, we evaluated genomic heterogeneity between HER2+ primary breast cancers and residual disease following neoadjuvant therapy. Results: There were 469 HER2+ early stage invasive breast cancer patients in the study cohort with a median follow-up of 6.0 years. The mean age at diagnosis was 53.6 (SD=13.4) years. 52.7% are European Americans and 39.2% are African Americans. 61.8% were hormone receptor-positive (HR+)/HER2+ and 37.7% were HR-/HER2+. Although patients undergoing neoadjuvant chemotherapy (n=191) had lager tumor and more nodal positive disease compared to patients undergoing adjuvant therapy (n=213), the two groups have similar recurrence-free survival rate (adjusted hazard ratio 0.88, 95% CI 0.50-1.55). In patients undergoing neoadjuvant therapy, 72 (37.7%) achieved pCR. Women with HR-/HER2+ tumors had higher pCR rate (58.9%), compared with 24.6% in women with HR+/HER2+ tumors (p<0.001). pCR was a very strong predictor for recurrence: the 5-year recurrence-free survival was 0.97 for patients with pCR in contrast to 0.75 for patients without pCR. Of the 38 patients whose tumor DNAs were sequenced to date, there were 23 primary tumors and 15 residual tumors. Compared to primary HER2+ tumors, residual tumors had lower frequency of somatic alterations in ERBB2 (33% vs. 74%, p=0.02), CDK12 (13% vs. 48%, p=0.039), and ATM (0% vs. 26%, p=0.063) compared to the primary tumors. The overall tumor mutational burden was 2.1 m/MB (range: 0.4-42.5) in primary tumors and 2.9 m/MB (0-12.5) in residual tumors (p=0.94). Median ERBB2 copy number is 10 (range: 2-20) in primary tumors and 3 (2-20) in residual tumors. Alterations in the CDK4 pathway (CDK4, CCND1, CDKN2A, and RB1) were more frequently detected among tumors with loss of ERBB2 amplification (50%) than tumors with ERBB2 amplification (10%, p=0.011), suggesting they may be mutually exclusive. In particular, 4 of the residual tumors had mutations in the CDK4 pathway and none co-occurred with ERBB2 amplification. Conclusions: These preliminary data support the importance of integrating genomic sequencing of residual tumors to identify potentially non-HER2 druggable targets as a post-neoadjuvant treatment strategy. Alternative approaches including addition of novel therapies such as CDK4 inhibitors may have the potential to reduce costs associated with extended use of HER2-targeted therapies when unlikely to improve overall survival for non-HER2 amplified tumors. Citation Format: Masaya Hattori, Dezheng Huo, Nike Beaubier, Padma Sheila Rajagopal, Toshio Yoshimatsu, Yonglan Zheng, Elisabeth Sveen, Galina Khramtsova, Fang Liu, Taylor Abboushi, Kevin White, Olufunmilayo Olopade. Comparative analysis of genomic landscape reveals heterogeneity in HER2-positive primary breast cancers and residual disease following neoadjuvant therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-10.

Genetic Aberrations in the CDK4 Pathway Are Associated with Innate Resistance to PD-1 Blockade in Chinese Patients with Non-Cutaneous Melanoma.

PD-1 checkpoint blockade immunotherapy induces long and durable response in patients with advanced melanoma. However, only a subset of patients with melanoma benefit from this approach. The mechanism triggering the innate resistance of anti-PD-1 therapy remains unclear.Experimental Design: Whole-exome sequencing (WES) and RNA sequencing (RNA-Seq) analyses were performed in a training cohort (n = 31) using baseline tumor biopsies of patients with advanced melanoma treated with the anti-PD-1 antibody. Copy-number variations (CNVs) for the genes CDK4, CCND1, and CDKN2A were assayed using a TaqMan copy-number assay in a validation cohort (n = 85). The effect of CDK4/6 inhibitors combined with anti-PD-1 antibody monotherapy was evaluated in PD-1-humanized mouse (C57BL/6-hPD-1) and humanized immune system (HIS) patient-derived xenograft (PDX) models. WES revealed several significant gene copy-number gains in the patients of no clinical benefit cohort, such as 12q14.1 loci, which harbor CDK4. The association between CDK4 gain and innate resistance to anti-PD-1 therapy was validated in 85 patients with melanoma (P < 0.05). RNA-Seq analysis of CDK4-normal cell lines and CDK4-normal tumors showed altered transcriptional output in TNFα signaling via NF-κB, inflammatory response, and IFNγ response gene set. In addition, CDK4/6 inhibitor (palbociclib) treatment increased PD-L1 protein levels and enhanced efficacy (P < 0.05) in the C57BL/6-hPD-1 melanoma cell and the HIS PDX model. In summary, we discovered that genetic aberrations in the CDK4 pathway are associated with innate resistance to anti-PD-1 therapy in patients with advanced melanoma. Moreover, our study provides a strong rationale for combining CDK4/6 inhibitors with anti-PD-1 antibody for the treatment of advanced melanomas.

Frequent genetic aberrations in the cell cycle related genes in mucosal melanoma indicate the potential for targeted therapy

BackgroundMelanoma is one of the most aggressive cancers with extremely poor prognosis, and the median survival time for stage IV patients is approximately 6 to 8 months. Unlike cutaneous melanoma, mucosal melanoma is a rare melanoma subtype among Caucasian patients but its incidence remains as high as 22.6% among Chinese patients. Screening specific genetic variations is the guideline to select targeted drugs for the treatment of advanced melanoma, whereas the genetic variation spectrum and potential therapeutic targets for mucosal melanoma are largely unclear. It is urgent to identify promising genetic variants for mucosal melanoma so as to develop effective targeted therapies for this disease.MethodsTumor samples from 213 Chinese mucosal melanoma patients were involved in this study. P16INK4a/Cyclin D1/CDK4 copy number was examined using the QuantiGene Plex DNA assay and the correlation between abnormal copy number and clinicopathological parameters was analyzed. Patient-derived xenograft models (PDX) were performed to detect the effects of CDK4/6 inhibitors on the proliferation of mucosal melanoma cells with altered copy number of CDK4 pathway (CDK4, Cyclin D1 and P16INK4a). The molecular mechanisms of CDK4/6 inhibitors on the proliferation of mucosal melanoma were analyzed by RNAseq.ResultsAmong the 213 samples, the amplification rate of CDK4 and CCND1 was 47.0% and 27.7%, respectively, and the deletion rate of P16INK4a was 57.7%. Patients with more than one genetic abnormality were up to 81.7%. CDK4 pathway gene copy number variation was not associated with the prognosis of patients with mucosal melanoma (P > 0.05). Drug sensitivity tests showed that AT7519, a broad-spectrum CDK inhibitor, and PD0332991, a specific CDK4/6 inhibitor, exhibited higher inhibitory effect on CDK4 signaling pathway abnormal mucosal melanoma cells-derived PDX tumors growth than CDK4 signaling pathway normal ones. RNA-seq analysis showed that CDK4 inhibitors may affect tumor proliferation through multiple signaling pathways.ConclusionsAbnormal copy number of cell cycle related genes is frequently found in mucosal melanoma. CDK4/6 inhibitors significantly suppress the PDX tumor growth with abnormal CDK4 pathway. CDK4 signaling variations predict the effectiveness of CDK4 inhibitors in mucosal melanoma.

Palbociclib (P) in advanced acral lentiginous melanoma (ALM) with CDK4 pathway gene aberrations.

9528 Background: Genetic Aberrations in the CDK4 Pathway occur in 82% of the ALM pts (Clin Cancer Res,2017, 23(22):6946-6957), which is predominant subtype in China. This study is to evaluate the anti-tumor activity of palbociclib, a CDK4/6 inhibitor, in advanced ALM pts with CDK pathway gene aberrations. Methods: In this phase II trial, patients with advanced ALM with CDK pathway gene aberrations (CDK4 or/and CCND1 amplification or/and CDKN2A loss) were treated with palbociclib 125 mg po, d1-21 of 28 day cycles. According to the study design, this trial will be closed if less than 2 patients yield objective response (OR) or stable disease (SD) at a preset 8-week landmark analysis. Secondary endpoints are progression-free survival (PFS), overall survival (OS) and safety. Results: Fifteen pts were enrolled from April to Nov 2018. All pts are included in the data analysis for demographics, safety, ORR, PFS and OS. Median age was 54 y (range, 25-74y), 6(40%) were males,5( 33.3%) had the primary sites of subungual, 4(26.7%) were of stage III. Median numbers of prior therapies: 2 (range: 0-5). Three (20.0%) pts achieved tumor shrinkage at 8 wks, including 1 confirmed partial responses (PR). Two pts had treatment ongoing at the data cutoff (January 2019). Median PFS was 2.5m (range, 1.5-8.8m), estimated mean OS was 8.1m (range, 6.7-9.4m). Four pts deceased due to disease progression. The most common treatment related adverse events (TRAEs) were leukopenia/neutropenia (87%; G3, 33%), thrombocytopenia (27%; G3, 7%), anemia (40%; G3, 7%). Conclusions: Monotherapy with palbociclib demonstrated preliminary activities in advanced melanoma pts with CDK pathway gene aberrations. Therefore, the study met its primary endpoint. Palbociclib had an acceptable safety profile as previous studies. Combination of CDK4/6 inhibitor with other agents is under consideration. Clinical trial information: NCT03454919.

β-hydroxy-β-methylbutyrate (HMB) improves mitochondrial function in myocytes through pathways involving PPARβ/δ and CDK4.

Mitochondrial dysfunction in skeletal muscle has emerged as key to the development of obesity and its related metabolic disorders. Leucine (Leu) is an essential amino acid that has been reported to increase mitochondrial biogenesis in muscle cells, as has its metabolite β-hydroxy-β-methylbutyrate (HMB). However, two questions-which one is more potent and what is the cellular mechanisms of the action of Leu and HMB-remain to be answered. Therefore we aimed to investigate the effects of Leu and HMB on mitochondrial function in C2 C12 myotubes and analyze the underlying molecular mechanism. The effects of Leu and HMB on mitochondrial mass, mitochondrial respiration capacity, and the expression of genes related to mitochondrial biogenesis were evaluated in C2 C12 myotubes. Differentiated myotubes were treated with Leu (0.5 mM) or HMB (50 μM) with or without PPARβ/δ antagonist (GSK3787, 1 μM) and CDK4 antagonist (LY2835219, 1.5 μM), respectively, for 24 h. The results indicated that treatment with Leu or HMB significantly increased mitochondrial mass, mitochondrial respiration capacity, and the messenger RNA expression of genes associated with mitochondrial biogenesis (P < 0.05). In addition, these positive effects of Leu or HMB on these parameters were attenuated by GSK3787 and LY2835219 treatments (P < 0.05). Our results provide evidence indicating that as with Leu, HMB alone could increase mitochondrial biogenesis and function via regulation of PPARβ/δ and CDK4 pathways. Moreover, HMB seems to be more potent than Leu in the positive regulation of mitochondrial biogenesis and function in C2 C12 myotubes because the dosage used for HMB was much lower than that for Leu.

Circular RNA hsa_circ_0016788 regulates hepatocellular carcinoma tumorigenesis through miR-486/CDK4 pathway.

Recent studies have revealed that circular RNAs (circRNAs) play important roles in the tumorigenesis of human cancer, including hepatocellular carcinoma (HCC). In present study, we screen the circular RNA expression profiles in HCC tissue and investigate the molecular roles on HCC tumorigenesis. Human circRNA microarray analysis showed there were total 1,245 differently expressed circular RNAs, including 756 up-regulated circRNAs and 489 down-regulated circRNAs, in three pairs of HCC tissue and adjacent normal tissue. Hsa_circ_0016788 was identified to be up-regulated in both HCC tissue and cell lines. Loss-of-functional experiments in vivo and vitro revealed that hsa_circ_0016788 silencing inhibited the proliferation, invasion and promoted the apoptosis in vitro, and inhibited the tumor growth in vivo. Bioinformatics tools and luciferase reporter assay validated that miR-486 targeted hsa_circ_0016788 and CDK4 accompanying with negatively correlated expression, suggesting the hsa_circ_0016788/miR-486/CDK4 pathway. Receiver operating characteristic (ROC) curve showed that hsa_circ_0016788 had high diagnostic value (AUC = 0.851). In summary, results reveal the role of hsa_circ_0016788/miR-486/CDK4 in HCC tumorigenesis, providing a novel therapeutic target for HCC.

Cell size sensing in animal cells coordinates anabolic growth rates and cell cycle progression to maintain cell size uniformity.

Cell size uniformity in healthy tissues suggests that control mechanisms might coordinate cell growth and division. We derived a method to assay whether cellular growth rates depend on cell size, by monitoring how variance in size changes as cells grow. Our data revealed that, twice during the cell cycle, growth rates are selectively increased in small cells and reduced in large cells, ensuring cell size uniformity. This regulation was also observed directly by monitoring nuclear growth in live cells. We also detected cell-size-dependent adjustments of G1 length, which further reduce variability. Combining our assays with chemical/genetic perturbations confirmed that cells employ two strategies, adjusting both cell cycle length and growth rate, to maintain the appropriate size. Additionally, although Rb signaling is not required for these regulatory behaviors, perturbing Cdk4 activity still influences cell size, suggesting that the Cdk4 pathway may play a role in designating the cell's target size.