Inhibition Of Cyclin Research Articles

Updates on the Anticancer Profile of Lycopene and its Probable Mechanism Against Breast and Gynecological Cancer

Abstract: Natural substances are gaining interest as anticancer agents nowadays due to the adverse effects of synthetic drugs. Among various natural substances, lycopene has emerged as a strong antioxidant agent and has been found to be effective against prostate, breast, colon, ovarian, liver, endometrial cancers, etc. This article reviews the therapeutic potential and proposed mechanism of action of lycopene against breast and gynecological cancer from 2005 to now. Experimental studies suggest that lycopene can inhibit tumor growth by regulating various signaling pathways for cell growth, arresting the cell cycle, and inducing cell apoptosis. Lycopene is reported to combat breast cancer specifically via mechanisms, such as regulation of expression of p53 and Bax, suppression of cyclin D, inhibiting the activation of ERK and Akt signaling pathway, and gynecological cancer via various signaling pathways such as STAT3, Nrf2, and NF- κB, down-regulation of ITGB1, ITGA5, FAK, MMP9, and EMT markers, etc.

Boronic Acid-Containing 3H- pyrazolo[4,3-f]quinoline Compounds as Dual CLK/ROCK Inhibitors with Anticancer Properties.

Background: The protein kinases CLK and ROCK play key roles in cell growth and migration, respectively, and are potential anticancer targets. ROCK inhibitors have been approved by the FDA for various diseases and CLK inhibitors are currently being trialed in the clinic as anticancer agents. Compounds with polypharmacology are desired, especially in oncology, due to the potential for high efficacy as well as addressing resistance issues. In this report, we have identified and characterized novel, boron-containing dual CLK/ROCK inhibitors with promising anticancer properties. Methods: A library of boronic acid-based CLK/ROCKi was synthesized via Povarov/Doebner-type multicomponent reactions. Kinase inhibition screening and cancer cell viability assays were performed to identify the hit compounds. To gain insights into the probable binding modes of the compounds to the kinases, docking studies were performed. Cell cycle analysis, qPCR and immunoblotting were carried out to further characterize the mode(s) of action of the lead candidates. Results: At 25 nM, the top compounds HSD1400 and HSD1791 inhibited CLK1 and 2 and ROCK2 at greater than 70%. While HSD1400 also inhibited CLK4, the C1 methylated analog HSD1791 did not inhibit CLK4. Antitumor effects of the top compounds were evaluated and dose-response analysis indicated potent inhibition of renal cancer and leukemia cell growth. Immunoblotting results indicated that the top compounds induce DNA damage via upregulation of p-H2AX. Moreover, flow cytometry results demonstrated that the top compounds promote cell cycle arrest in the renal cancer cell line, Caki-1. qPCR and immunoblotting analysis upon HSD1791 dosing indicated suppression of cyclin D/Rb oncogenic pathway upon compound treatment. Conclusions: Novel boronic acid-containing pyrazolo[4,3-f]quinoline-based dual CLK/ROCK inhibitors were identified. The so-called "magic methylation" design approach was used to tune CLK selectivity. Additionally, the findings demonstrate potent in vitro anticancer activity of the lead candidates against renal cancer and leukemia. This adds to the growing list of boron-containing compounds that display biological activities.

Fenbendazole and Diisopropylamine Dichloroacetate Exert Synergistic Anti-cancer Effects by Inducing Apoptosis and Arresting the Cell Cycle in A549 Lung Cancer Cells.

Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for approximately 2 million new cases and 1.8 million deaths annually. Standard treatment options include surgery, radiation therapy, chemotherapy, and targeted therapies. Despite advancements over the past 25 years, the prognosis of patients with lung cancer remains poor. This study evaluated the synergistic anticancer effects of fenbendazole (FZ) and diisopropylamine dichloroacetate (DADA) on A549 lung cancer cells. Fenbendazole (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl) carbamate) is a broad-spectrum benzimidazole anthelmintic commonly used in veterinary medicine. Diisopropylamine Dichloroacetate (DADA), an over-the-counter treatment for chronic liver disease, has demonstrated anti-tumor properties as an inhibitor of pyruvate dehydrogenase kinase. The combination of FZ and DADA exhibited a synergistic effect on inhibiting the proliferation of A549 lung cancer cells. After 48 h of treatment, the FZ-DADA combination produced reactive oxygen species (ROS) and promoted apoptosis by down-regulating Bcl2 and up-regulating BAX protein expression. The combination activated caspase-3, caspase-7, and PARP, further driving apoptosis in A549 cells. The FZ-DADA treatment also induced cell cycle arrest, as evidenced by the inhibition of Cyclin A and Cyclin E proteins. The synergistic anticancer effects of the FZ-DADA combination were confirmed at both cellular and protein levels in A549 lung cancer cells. The combination modulates key apoptotic proteins, induces cell cycle arrest, and increases mitochondrial ROS production, suggesting a promising approach for lung cancer treatment that warrants further investigation and development.

Uterine fibroid metastasis as the initial manifestation of lobular breast cancer

The occurrence of breast cancer metastasis in a uterine leiomyoma is exceptional, and its diagnosis prior to detection of the primary tumor is even rarer. We describe at case of a 52-year-old female patient with a large fast growing uterine fibroid that caused pelvic pain and abdominal distention. A total abdominal hysterectomy with bilateral salpingo-oophorectomy was performed. Histopathology of the surgical specimen showed a characteristic pattern of invasive breast lobular carcinoma in the leiomyoma. A magnetic resonance image of the breast was then performed, which reveled primary and bilateral breast cancer. She subsequently underwent neoadjuvant endocrine therapy plus cyclin inhibitors, breast-conserving surgery, and radiotherapy. To date, the patient is alive and asymptomatic after 3 years of follow-up.

Multiple outcomes of the germline p16INK4a mutation affecting senescence and immunity in human skin.

The integrated behaviour of multiple senescent cell types within a single human tissue leading to the development of malignancy is unclear. Patients with Familial Melanoma Syndrome (FMS) have heterozygous germline defects in the CDKN2A gene coding for the cyclin inhibitor p16INK4a. Melanocytes within skin biopsies from FMS patients express significantly less p16INK4a but express higher levels of the DNA-damage protein 𝛾H2AX a than fibroblastic cells. However, patient fibroblasts also exhibit defects since senescent cells do not increase in the skin during ageing and fibroblasts isolated from the skin of patients have increased replicative capacity compared to control fibroblasts invitro, culminating in abnormal nuclear morphology. Patient derived fibroblasts also secreted less SASP than control cells. Predisposition of FMS patients to melanoma may therefore result from integrated dysregulation of senescence in multiple cell types invivo. The inherently greater levels of DNA damage and the overdependence of melanocytes on p16 for cell cycle inhibition after DNA damage makes them exquisitely susceptible to malignant transformation. This may be accentuated by senescence-related defects in fibroblasts, in particular reduced SASP secretion that hinders recruitment of T cells in the steady state and thus reduces cutaneous immunosurveillance invivo.

Cucurbitacin: Unveiling its Role as Phytomolecule in Health Benefits

Abstract: In recent scientific studies, a variety of phytochemicals, including carotenoids, polyphenols, isoprenoids, phytosterols, saponins, and dietetic fibers, besides polysaccharides, have been linked to beneficial health effects, which include reducing the risk of diabetes, obesity, and cancer, cardiovascular diseases, and other conditions. Squash, pumpkin, cucumber, and melons are just a few examples of the Cucurbitaceae family plants that contain cucurbitacins, highly oxygenated tetracyclic triterpenes. Since ancient times, various traditional remedies have been made from plants that contain cucurbitacins. Several cucurbitacins (A, B, C, D, E, F, G, I, J, K, L, O, P, Q, R, S) have an extensive assortment of bioactivities, including hepatoprotective activity, liver protection, anticancer, anti-inflammatory, antiviral, and anti-diabetic properties. Cucurbitacins, for instance, have a well-known anticancer effect. Specific reports suggest that cucurbitacins stimulate apoptosis via the JAK/STAT3 pathway. Through cyclin inhibition, cucurbitacins may inhibit the cell cycle. Besides, they expedite autophagy while preventing cancer cells from migrating and infecting other tissues. Cucurbitacins have been categorized as signal transducers and activators of transcription. It is soundly acknowledged that the plant-based compound cucurbitacin B (CuB) has insecticidal and repellent properties. CuB has been shown to have non-specific toxicity and limited bioavailability in studies evaluating its toxicity and pharmacokinetic characteristics. Hence, we aimed to concentrate on the numerous properties of cucurbitacins in this article, including their chemistry, an analysis of biosynthesis, several types of bioactivities, and studies on their toxicity.

How overall survival is conditioned in the progression to treatment with cyclin inhibitors plus endocrine treatment in metastatic breast cancer with luminal phenotype.

e13079 Background: CDK4/6 inhibitors plus endocrinotherapy (ET) is the standard first-line treatment for metastatic breast cancer with a luminal phenotype. However, the response to this treatment is highly dependent on its previous efficacy. Methods: We analyzed the overall survival in a cohort of 127 patients with metastatic luminal breast cancer in progression after treatment with CDK4/6 inhibitors plus ET. Results: The median age of the group was 67 years (41-96), the interval time between initial diagnosis and first recurrence was 0 in 41 (32%), 1-60 months in 37 (29%) and more than 60 months in 39 (38%). The first line in the metastatic setting was CDK4/6 plus ET in 81 patients (64%), ET in 15 (12%) and 31 (24%) were treated with chemotherapy. The status of HER2 expression was HER2 low in 77% and 43 % had visceral metastases disease. The median progression-free survival for the patients treated with CDK4/6 inhibitors plus ET was 9 months, with a total of deaths of 69 (55%). Overall survival after progression on CDK4/6 plus ET was 38 months (28 – 47) and the most important factor for overall survival was the response time to treatment with CDK4/6 inhibitors plus ET. Conclusions: The most important factor for overall survival is the duration of treatment with CDK4/6 inhibitors plus ET. Patients with a response time of less than 6 months had a median overall survival after progression of 11 months (3 – 19), while the 6- to 12-month group had a median of 39 months (19 – 59) and the group with more than 12 months had the best prognosis with a median overall survival of 53 months (37 – 68). We need to improve the new treatment options for patients who respond for less than 6 months to treatment with CDK4/6 plus ET.

Abstract PO3-20-02: Olaparib front line therapy with endocrine therapy in locally advanced unresectable or in metastatic breast cancer: OFELIA TRIAL

Abstract INTRODUCTION: Currently, patients with recurrent or metastatic hormone-sensitive breast cancer are treated with endocrine therapy (ET) in combination with cyclin inhibitors, as it has been shown to improve both overall survival and progression-free survival. However, it has been observed that patients with gBRCA1 or gBRCA2 mutations, who receive treatment with endocrine therapy and CDK4/6 inhibitors, have lower OS and lower PFS compared to those without mutations. These findings could be due to the presence of higher cyclin E mRNA levels, which encode the protein that stimulates CDK2 activity provoking a resistance to CDK4/6 inhibitors. Having this said, the use of PARP inhibitors a potential first-line therapeutic option in combination with endocrine therapy could be an option to be re evaluated. JUSTIFICATION: PARP inhibitors (iPARP) are recommended in international guidelines as second-line palliative treatment and also as adjuvant management in the case of Olaparib. The use of iPARP is approved only as a second-line treatment in HER2 hormone-sensitive breast cancer, according to the results of the EMBRACA and OlympiAD clinical trials. However, these two clinical trials were not designed to evaluate its effectiveness as first-line palliative treatment. Given that patients with gBRCA mutations have lower overall survival when administered cyclin inhibitors (iCDK 4/6) + Endocrine therapy (ET) compared to those without mutations, it is necessary to reevaluate the use of iPARP as first-line palliative treatment in conjunction with ET, with the expectation of achieving a similar objective response rate and progression-free survival. OBJECTIVE: To Determine the ORR in patients with recurrent or metastatic hormone-sensitive HER2-negative breast cancer managed with PARP inhibitors + Endocrine therapy. HYPOTHESIS: The use of PARP inhibitors in combination with endocrine therapy will have an objective response rate of 60% or higher in both early stages and advanced disease. METHODOLOGY: Phase 2 open-label, multicenter clinical trial. Study Population: Cohort : All pre- or postmenopausal patients with a diagnosis of recurrent or metastatic hormone-sensitive HER2-negative breast cancer with gBRCA1 and/or gBRCA2 mutation. Sample: The inclusion of patients will be based on meeting the inclusion criteria. We plan to achieve an ORR of at least 60% with an acceptable minimum of 40%. Assuming a one-sided alpha level of 0.05 and statistical power of 80%, a two-stage Simon design was used to calculate the required sample size. In the first stage, a minimum of 23 patients is assumed, of which 14 patients need to achieve the desired response rate to proceed to the second stage. In the second stage, an additional 28 patients will be added to obtain a total sample size of 51 patients. The objective response rate will be evaluated using RECIST v1.1 criteria at 8 weeks after initiating treatment, with continued assessment during the first year, followed by assessments every 12 weeks during the second and third year, and then every 24 weeks until progression or toxicity. EXPECTED RESULTS: PARP inhibitors + Endocrine therapy will have a similar ORR as reported with cyclin inhibitors + Endocrine therapy, as well as a benefit in progression-free survival. Citation Format: Carlos Arturo González Núñez, Paula Cabrera-Galeana, Juan Enrique Bargalló Rocha, Rafael Vazquez-Romo, Sergio Aguilar-Villanueva, Alexandra Garcilazo, Areli Velazquez-Martinez, Andrea Maliachi, Ruben Rodriguez. Olaparib front line therapy with endocrine therapy in locally advanced unresectable or in metastatic breast cancer: OFELIA TRIAL [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-20-02.

Abstract PO2-01-10: 21-Gene Recurrence Score Index (ONCOTYPE DX) as a predictive biomarker for neoadjuvant chemotherapy response and outcome in patients with HR+HER2- breast cancer

Abstract Background: Hormone receptor positive (HR+), HER2- early stage breast cancer (EBC) has shown lower response to neoadjuvant chemotherapy (NCT) compared with other clinicopathologic subtypes. ONCOTYPE DX may help inform systemic treatment decisions for EBC and predicting likelihood of pathological complete response (pCR) or chemosensitivity. We analyze the pathologic response according ONCOTYPE RS and their prognostic value. Methods: We included a serie of 139 consecutive patients with high clinical risk factor with criteria to received NCT than have performed an ONCOTYPE DX test previous to start NCT. The median age was 51 years with 65 patients (47%) less than 50 years. Median initial tumoral size was 25 mm (9-97), T1: 35%, T2: 51% and T3: 14%. 70 patients (50%) have initial axillary node involvement. Median ONCOTYPE DX score was 29 (12 – 76) , RS < 25 in 36% and RS < 30 in 54%. Complete pathologic response (pCR) was found in 35 patients (25%), and by RCB was: RCB:0 23%, RCB-1 18%, RCB-2 24% and RCB-3 34%. Results: pCR score was correlated with ONCOTYPE DX (pearson : p:0,001) , patients with ONCOTYPE RS score superior to 30 has a total of 37% of pCR in contrast to a 14% in patients with ONCOTYPE RS score less than 30. Median follow-up was 32 months (5 -100). The rate of recurrence was similar independently the ONCOTYPE RS score (12% < 30 ; 15% >30) , but patients with ONCOTYPE RS < 30 with achieve pCR had better prognostic compared to patients that not achieve pCR (0% vs 14%). Patients with ONCOTYPE RS > 30 tumors that had a pCR the recurrence rate was 8% compared to 20% in patients with non pCR. Conclusions: These data suggest ONCOTYPE DX predicts pCR in HR+HER2- BC patients, especially in patients with a RS superior to 30 with an high rate of pCR of 37%. Patients with ONCOTYPE DX less than 30 that achieved pCR had the better outcomes and patients with ONCOTYPE DX superior to 30 that not achieved pCR had the worst outcomes with a total of 20% of recurrence. ONCOTYPE DX predicts the possibility of achieve a pCR and patients with a high ONCOTYPE DX RS score with residual disease have a high recurrence despite receiving treatment with chemotherapy, which requires a better adjuvant treatment, possibly with CDK 4/6 cyclin inhibitors. Citation Format: Ariadna Gasol Cudós, Serafin Morales Murillo, Noemí Tuset Der-Abrain, Felip Vilardell Villellas, Laura Arbones Cid, Jordi Melé Olivé, Carles Canosa Morales. 21-Gene Recurrence Score Index (ONCOTYPE DX) as a predictive biomarker for neoadjuvant chemotherapy response and outcome in patients with HR+HER2- breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-01-10.

Abstract PO5-05-01: ASSESING THE CLINICAL AND SURVIVAL RESULTS OF PATIENTS WITH HR-POSITIVE, HER2 NEGATIVE ADVANCED BREAST CANCER TREATED WITH CDK 4/6 INHIBITORS IN A SPANISH COHORT

Abstract BACKGROUND First line treatment of HR-positive, HER2 negative advanced breast cancer is based on endocrine therapy plus CDK 4/6 inhibitors (CDK 4/6i). Recently, there has been published in ASCO 2023 results from SONIA trial, which explores the use of CDK 4/6i in second-line treatment after monotherapy with endocrine therapy. This trial have shown using a CDK4/6i in the first-line setting did not significantly prolong the time from random assignment to progression on second-line therapy (PFS2) or overall survival (OS) when compared with deferred use until the second-line setting. METHODS We conducted a descriptive, observational and retrospective analyses of patients treated in our centre with CDK4/6i, comparing demographic, clinical and survival results. We also analysed different outcomes between first versus second line CDK4/6i. RESULTS A cohort of 153 patients were included, treated between January 2018 and June 2023, with a median age of 50 years. At diagnosis, 49% were stage I-II and 31% stage IV. 59% had visceral metastases at the start of CDK4/6i treatment. Palbociclib was the most commonly CDK4/6i used (55%), followed by Ribociclib (32%) and Abemaciclib (12.5%). The most widely endocrine therapy used was letrozole (60%), followed by fulvestrant (38%). 58% received CDK4/6i in first-line setting while 9% received them in second-line. The overall median progression-free survival (mPFS) was 18 months. In a subgroup analysis, the mPFS was 19 months with Palbociclib, 26 months with Ribociclib, and 31 months with Abemaciclib. Baseline characteristics were similar between subgroups of CDK4/6i. Better survival data were obtained with letrozole than with fulvestrant (not reached versus 29 months OS respectively). After CDK4/6i disease progression the most common subsequent treatment was chemotherapy (capecitabine, 23%). The median OS with palbociclib and abemaciclib was 37 months, while not reached with ribociclib. We analysed PFS2 using CDK4/6i in the first-line setting compared with deferred use until the second-line. The use of CDK4/6i in second-line showed a PFS2 of 45 months, while first-line PFS2 was 26 months. CONCLUSIONS Our study results are consistent with previous studies assesing the efficacy of cyclin inhibitors in first and second-line setting. Palbociclib seems to have a lower PFS rate. Patients treated with letrozole had a stadistically significant benefit in survival outcomes. Our data continue the trend observed in the SONIA trial in terms of PFS2. Using endocrine therapy as first-line monotherapy may be a valid treatment option. However, larger sample sizes are required to draw definitive conclusions. The identification of potential predictors of treatment response may help in the selection of the most effective treatment for patients with HR-positive HER2 negative advanced breast cancer. Citation Format: Alicia Cano-Jimenez, Rocio Urbano-Cubero, Ruben Garcia-Muñoz, Ana Maria Jaen-Morago, Maria Lomas-Garrido, Pedro Sánchez-Rovira. ASSESING THE CLINICAL AND SURVIVAL RESULTS OF PATIENTS WITH HR-POSITIVE, HER2 NEGATIVE ADVANCED BREAST CANCER TREATED WITH CDK 4/6 INHIBITORS IN A SPANISH COHORT [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-01.

Abstract 3077: Identifying Regulators Of Human Adventitial Fibroblast Activation Through A Genome-wide CRISPR Screen

Background: Vascular diseases, including atherosclerosis, are the leading cause of death in the US and world-wide. A quiescent population of fibroblasts that reside in the adventitia (AdvFib) activates and expands upon different vascular insults and contribute to the progression of vascular diseases. However, what regulates AdvFib’s activation remains unknown. We designed a genome-wide CRISPRi screen on human AdvFib to identify these regulators. Methods: We generated an immortalized human coronary AdvFib line that stably expresses dCas9-KRAB, which effectively silences any genes of interest. These cells were infected with a pool of lentivirus carrying guide RNAs (gRNA) covering the entire transcriptome at 200X coverage and allowed to expand freely. Frequency of gRNAs was assessed at different cell passages to identify those that influence AdvFib activation and was followed by experimental validation. Lastly, bioinformatic analysis was performed to determine regulatory pathways and gene networks involved in AdvFib activation. Results: Comparing relative gRNA abundances from early to late passages revealed several hundred putative regulators of AdvFib activation. GO biological process analysis of the top 250 enriched gRNA targets demonstrated disproportionate representation of cyclin inhibitors, validating the effectiveness of our screening. Molecular signatures for adipogenesis and key regulators of tissue mineralization including IBSP and ENPP1 were notably enriched in our screen. Human disease association using PhenGenI revealed a significant association with atherosclerosis. Indeed, numerous coronary artery disease GWAS genes including TCF21 , ZEB2 , CDKN1A , and CDKN2A were enriched in our screen, providing additional support for AdvFib’s importance in atherosclerosis. These hits were subsequently validated individually in vitro. Conclusion: A genome-wide CRISPRi screen of AdvFib activation modulators identified numerous potential regulator genes, many of which have known genetic and observational association with human vascular diseases. This study affirms the potentially important role of AdvFib in human vascular disease and may pave the way to future therapeutics targeting this often neglected cell population.

Management of cutaneous adverse events caused by antineoplastic therapies: a single-center experience.

Cutaneous adverse events can occur in patients treated with antineoplastic treatments, albeit their incidence has not been defined yet. The clinical presentation of CAEs related to anticancer treatments can vary. The purpose of our study is to characterize skin toxicities during oncological treatments, manage such adverse events to improve patients' quality of life, and ensure therapeutic adherence. We conducted a single-center prospective study which provided the enrollment of all patients referred to the Skin Toxicity Outpatient Clinic for the occurrence of cutaneous adverse events secondary to an ongoing antineoplastic treatment, between July 2021 and June 2023. We analyzed clinical features, and we described our therapeutic approach. Based on the type of drug assumed, chemotherapy-induced skin toxicity in 24 (38.7%) of the 62 evaluated patients, target therapies in 18 (29.0%), CDK4/6 cyclin inhibitors in 12 (19.4%), and immunotherapy in 6 (9.7%), while skin adverse events secondary to hormone therapy were seen in two patients. The most common cutaneous adverse event in our experience was rosaceiform rash of the face, followed by eczematous rash, hand-foot syndrome, and folliculitis. The present study is aimed at describing the variability and heterogeneity of clinical manifestations of different pharmacological classes used in oncological patients, as well as the different pathogenesis of skin damage. Chemotherapy very frequently causes skin toxicities that are often underestimated by clinicians. Their adequate recognition and optimal treatment lead to total recovery and allow better adhesion to chemotherapy.

A new treatment for breast cancer using a combination of two drugs: AZD9496 and palbociclib

In this study, we examined a mathematical model of breast cancer (BC) treatment that combines an oral oestrogen receptor inhibitor, AZD9496 with Palbociclib, a selective inhibitor of cyclin- dependent kinases CDK4 and CDK6. Treatment is described by analytical functions that enable us to control the dosage and time interval of the treatment, thus personalising the treatment for each patient. Initially, we investigated the effect of each treatment separately, and finally, we investigated the combination of both treatments. By applying numerical simulations, we confirmed that the combination of AZD9496 with palbociclib was the optimal treatment for BC. The dosage of AZD9496 increased and decreased throughout the treatment period, while the intervals were constant between treatments. Palbociclib changed almost cyclically, whereas the time intervals remained constant. To investigate the mathematical model, we applied the singularly perturbed homotopy analysis method, which is a numerical algorithm. The significant advantage of this method is that the mathematical model does not have to contain a small parameter (as is standard in perturbation theory). However, it is possible to artificially introduce a small parameter into the system of equations, making it possible to study the model using asymptotic methods.

Harnessing Btki Therapy By CDK4/6i Control of T Effector Memory Cells and T Cell Surveillance in Mantle Cell Lymphoma

Drug resistance remains a formidable challenge in mantle cell lymphoma (MCL). Cell cycle dysregulation driven by aberrant Cyclin D1 and CDK4 expression is a hallmark for MCL, providing a rationale for targeting the cell cycle in MCL therapy. We have demonstrated in preclinical studies that inhibition of CDK4/6 not only induces early G1 cell cycle arrest, but also reprograms MCL cells for cytotoxic killing. On this basis, we investigated if inhibition of cyclin D1/CDK4 with palbociclib (CDK4/6 inhibitor) could reprogram recurrent MCL to deepen and prolong the ibrutinib (BTK inhibitor) response in a phase I clinical trial, and the resistance mechanism by longitudinal genomic analysis of sequential samples from individual patients in the context of the clinical response. Palbociclib was administered to MCL patients on days 1-21 of a 28-day cycle; ibrutinib was given continuously. For longitudinal genomic analysis, sequential tissue and blood specimens from 27 evaluable MCL patients before and during therapy, and on progression were collected. Single cell RNA-seq (scRNA-seq) of PBMC or the monocytic fractions from bone marrow and lymph node (53 samples) was performed using a unique in house MCL cell-specific library. PBMC from normal donors (n=4) and untreated MCL patients (n=4) served as controls. The data were then subject to multiplex analysis with whole transcriptome sequencing and whole exome sequencing of MCL cells isolated from the same specimens, as well as flowcytometry in conjunction with IHC. Total numbers of MCL and immune cells in each specimen were deduced by integrating complete blood counts (CBC) with differential with scRNA-seq or flowcytometry. CDK4/6 Inhibition appears to deepen and prolong the BTKi response, with a CR rate of 42% and 5 patients (2 CR and 3 PR) remained on therapy for ~ 9 years. Longitudinal scRNA-seq (210,000 cells) revealed that MCL cells comprise 4 major transcriptomically distinct clusters. Cluster 1 (C1) resembles quiescent normal B cells; C2 mimics hyper-activated B cells that are enriched for signatures of BCR and cytokine signaling; C3 represents non-proliferating, long-lived MCL cells; and C4 is highly proliferative. RNA velocity analysis further indicates that having progressed to late G1 after evading CDK4/6 inhibition, MCL cells in C2 are in transit to C4. By contrast, C3 is a terminal node of MCL cells arrested in late G1 with elevated BCL2 expression Primary resistance and progression on therapy were associated with a marked expansion of either long-live non-proliferating C3 MCL cells, or C2 MCL cells that fuel the proliferating C4 MCL cells. Resistance was also associated with a profound reduction in MHC I and MHC II expression on MCL cells in some patients. Moreover, concurrent with disease progression, CD8+ T and CD+T cells were rapidly depleted by different mechanisms ( Figure 1). CD8+ T cells were proliferating and eliminated by exhaustion, as evidenced by the marked expression of LAG3 TIGIT and TIM3, and a failure to maintain CD8+ T effector memory cells. By contrast, CD4+ T cells were not proliferating and appeared die from glucose starvation. Collectively, these data suggest that T cell maintenance and surveillance is pivotal in sustaining a durable clinical response to combined inhibition of CDK4/6 and BTK. Guided by these discoveries, we have now restored BTKi sensitivity ex vivo in MCL cells from resistant patientsby targeting patient-specific expansion of C2 or C3 in vivo. In one resistant patient, this led to the maintenance of CD4+ and CD8+ T effector memory cells and a CR in response to venetoclax plus BTKi for nearly 3 years and continuing. In summary, by integrated longitudinal scRNA-seq analysis of a hypothesis-driven therapy, we have provided the first evidence that 1) MCL cells comprise 4 major transcriptomically distinct clusters; 2) resistance to CDK4/6i is associated with expansion of C2 that fuels the proliferating C4 MCL cells, or the long-lived C3 MCL cells with elevated BCL2 expression; 3) CDK4/6i harnesses BTKi response by promoting the maintenance of effector memory T cells; 4) combined inhibition of BCL2 and BTK overrides resistance to CDK4/6i and BTKi, leading to a durable clinical response; and 5) T cell surveillance is pivotal for a durable response to CDK4/6, BTK or BCL2 inhibition; implicating genome-guided combination therapy to overcome therapy resistance in MCL.

Denosumab and Zoledronic Acid Differently Affect Circulating Immune Subsets: A Possible Role in the Onset of MRONJ.

This work investigated whether the anti-resorptive drugs (ARDs) zoledronic acid (Zol) and denosumab (Dmab) affect differently the levels of circulating immune cell subsets, possibly predicting the risk of developing medication-related ONJ (MRONJ) during the first 18 months of treatment. Blood samples were collected from 10 bone metastatic breast cancer patients receiving cyclin inhibitors at 0, 6, 12, and 18 months from the beginning of Dmab or Zol treatment. Eight breast cancer patients already diagnosed with MRONJ and treated with cyclin inhibitors and ARDs were in the control group. PBMCs were isolated; the trend of circulating immune subsets during the ARD treatment was monitored, and 12 pro-inflammatory cytokines were analyzed in sera using flow cytometry. In Dmab-treated patients, activated T cells were stable or increased, as were the levels of IL-12, TNF-α, GM-CSF, IL-5, and IL-10, sustaining them. In Zol-treated patients, CD8+T cells decreased, and the level of IFN-γ was undetectable. γδT cells were not altered in Dmab-treated patients, while they dramatically decreased in Zol-treated patients. In the MRONJ control group, Zol-ONJ patients showed a reduction in activated T cells and γδT cells compared to Dmab-ONJ patients. Dmab was less immunosuppressive than Zol, not affecting γδT cells and increasing activated T cells.

Fundamental Considerations of Targeted Drug Therapies for Breast Cancer

Breast cancer is a complex disease for which pharmacological treatment does not guarantee success or cure. In addition, current pharmacological therapies induce unwanted side effects due to their lack of specificity or selectivity. Therefore, it is necessary to develop new therapeutic options to improve these aspects. Currently, phytochemicals with antineoplastic properties have been identified from a wide variety of plant sources, and new therapeutic options have been developed based on the conjugation of drugs with polymeric matrices, resulting in nanoparticles or hydrogels with improved properties. Some antineoplastic drugs have been conjugated with antibodies to improve their selectivity and specificity. One of the most important advances in the treatment of breast cancer has been the development of cyclin inhibitors and gene therapy. This review provides an overview of drugs derived from medicinal plants and polymeric matrices with high potential for use in the treatment of breast cancer. We also highlight the clinical evidence for the use of anti-HER2 monoclonal antibodies and cyclin inhibitors in breast cancer, as well as the advantages of using conjugated antibodies. Finally, we mention some considerations that should be taken into account in the search for new therapeutic agents from phytochemicals, polymers, antibodies, cyclin inhibitors, and gene therapy focused on the treatment of breast cancer.

HARNESSING BTKI THERAPY BY CDK4/6I CONTROL OF T CELL SURVEILLANCE

Introduction: Drug resistance remains a formidable challenge in mantle cell lymphoma (MCL). Cell cycle dysregulation driven by aberrant Cyclin D1 and CDK4 expression is a hallmark for MCL. By inhibition of CDK4/6, we have developed a novel strategy that both inhibits proliferation of MCL cells and reprograms them for cytotoxic killing. In a phase I clinical trial we investigated if inhibition of cyclin D1/CDK4 with palbociclib could reprogram recurrent MCL to deepen and prolong the ibrutinib response, and the mechanism for resistance by longitudinal genomic analysis of sequential samples from individual patients in the context of the clinical response. Methods: Palbociclib was administered to recurrent MCL patients on days 1–21 of a 28-day cycle; ibrutinib was given continuously. For longitudinal genomic analysis, sequential tissue and blood specimens from 27 evaluable MCL patients before, during therapy and on progression were collected. Single cell RNA-seq (scRNA-seq) of PBMC or the monocytic fractions from bone marrow and lymph node was performed using a unique in house MCL-specific library. The data were then subject to multiplex analysis with whole transcriptome sequencing and whole exome sequencing of purified MCL cells and flowcytometry of the same samples in conjunction with IHC. Results: Palbociclib appears to deepen and prolong the ibrutinib response; the CR rate was 42% CR, and 5 patients (2 CR and 3 PR) remained on therapy for >8 years. Longitudinal scRNA-seq further revealed that MCL cells comprise 4 major transcriptomically distinct clusters (C)s. Primary resistance and progression on therapy were associated with a marked expansion of either long-live non-proliferating C3 cells or C2 cells that fuel the proliferating C4 cells. Resistance was associated with a profound reduction in MHC I and MHC II expression and disease progression was further accompanied by a rapid loss of both CD8+ and CD+ T cells. This suggests T cell surveillance plays a critical role in maintaining a durable ibrutinib response. Guided by our discoveries, we have restored ibrutinib sensitivity by target patient-specific expansion of C2 or C3 MCL cells ex vivo. In one patient, this led to restoration of CD4+ and CD8+T cells and a CR over 2.5 years. Conclusion: T cell surveillance plays a critical role in prolonging the ibrutinib response by CDK4/6 inhibition, implicating genome-guided combination therapy to overcome ibrutinib resistance in MCL. The research was funded by: PO1 grant from the National Cancer Institute MCL-RI grant from Leukemia and Lymphoma Society Keywords: combination therapies, genomics, epigenomics, and other-omics, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Antitumor effects of erlotinib in combination with berberine in A431 cells

BackgroundFirst-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, have been shown to target tumors with L858R (exon 21) and exon 19 deletions, resulting in significant clinical benefits. However, acquired resistance often occurs due to EGFR mutations. Therefore, novel therapeutic strategies for treatment of patients with EGFR-positive tumors are needed. Berberine (BBR) is an active alkaloid extracted from pharmaceutical plants such as Coptis chinensis. Berberine has been shown to significantly inhibit EGFR activity and mediate anticancer effects in multiple preclinical studies. We investigated whether combining BBR with erlotinib could augment erlotinib-induced cell growth inhibition of EGFR-positive cells in a mouse xenograft model.MethodsWe examined the antitumor activities and potential mechanisms of erlotinib in combination with berberine in vitro and in vivo using the MTT assay, immunoblotting, flow cytometry, and tumor xenograft models.ResultsIn vitro studies with A431 cells showed that synergistic cell growth inhibition by the combination of BBR and erlotinib was associated with significantly greater inhibition of pEGFR and pAKT, and inhibition of cyclin D and Bcl-2 expression compared to that observed in response to BBR or erlotinib alone. The efficacy of the combination treatment was also investigated in nude mice. Consistent with the in vitro results, BBR plus erlotinib significantly reduced tumor growth.ConclusionOur data supported use of BBR in combination with erlotinib as a novel strategy for treatment of patients with EGFR positive tumors.

Daidzein and Chicory Extract Arrest the Cell Cycle via Inhibition of Cyclin D/CDK4 and Cyclin A/CDK2 Gene Expression in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers, associated with a high rate of mortality. A disturbance between cell proliferation and cell death is one of the cancer hallmarks including HCC. Cell proliferation is mainly controlled by the cell cycle. The arrest of the cell cycle is one of the important targets of anticancer agents. The present study tries to clarify the exact role of some natural products such as daidzein (DAZ) and alcoholic chicory leaf extract (CE), as possible regulators of cell cycle and apoptosis. HCC in rats was induced using diethylnitrosamine (DENA). Ninety rats were allocated and divided equally into nine groups, treated with CE, DAZ, a combination of both, and sorafenib with non-treated control groups. Treatment with CE, DAZ, and their combination significantly downregulated hepatic tissue expression of cyclin D1/CDK4 axis as well as cyclin A/CDK2 axis. The suggested therapeutic protocol inhibited the proliferation and dampened Bcl-2 expression. Furthermore, the efficiency of combining CE and DAZ demonstrated a potency comparable to sorafenib in terms of cyclin D/CDK4 axis expression, as well as; this combination protocol was more potent in revealing a potentiated inhibitory effect on cyclin A and Ki-67 expression. Treatment with DAZ or CE alone, or in combination, could possess an inhibitory effect on hepatocarcinogenesis via cell cycle arrest, inhibition of proliferation through suppression of Ki-67 expression, and apoptosis induction, mediated by downregulation of Bcl-2.

247P Efficacy of therapy directed against PI3K and cyclin inhibitors based on the location of PI3K mutations

Activation of the phosphatidylinositol-3-kinase (PI3K) pathway is responsible of activating the phosphorylation cascade that regulates cell survival and metabolism. Mutations in this pathway may occur in 28-46% of HR+/HER2- advanced breast cancers. It is known that the presence of this mutation confers a worse prognosis. We set out to analyze if PI3K mutations in different exons confer resistance to treatments directed against this target and to cyclin inhibitors (inh-CDK4/6). We have analyzed 96 patients with advanced breast cancer HR+/HER2- subsidiaries of treatment with PI3K-targeted therapy in the hospital of Jaén from February/20 to January/23. To detect PIK3CA mutations we used the cobas ® diagnostic kit that detects: exons 1 (R88Q), 4, 7 (C420R), 9 (E542K, E545K/A/D/G, Q546E/R) and 20 (H1047R/Y/L). We obtained 26% mutations in PI3K (5% exon 1, 40% exon 9, 55% exon 20). All in women with a mean age at diagnosis of 48 years and 56 at diagnosis of metastases, located at bone 65%, lung 35%, liver 23% and CNS 4% All patients have been treated with inh-CDK4/6. The median progression-free survival (mSLP) is 17 months (1-60). In patients with exon 1 mutation, mSLP is 25 months, in exon 9, 13 (2-43) and in exon 20, 19 (1-60) 60% of the patients have progressed to inh-CDK4/6 and as next treatment we used alpelisib. In 58% corresponds to 2 line in metastatic disease. The mSLP is 5 months (1-22) with a median follow-up of 14 months (1-34) and 69% of events. In patients with mutation in exon 1 the mSLP is 2 months, in exon 9, 5 (1-13) and in exon 20, 9 (2-22). Treatment was associated with an antiestrogen (77%) with a mSLP 7 months (1-22) or an aromatase inhibitor with a mSLP 3 months (2-10). 53% suffered grade 3 adverse effects (G3 AE): hyperglycemia (80% exon 9, 16% exon 20), diarrhea (20% exon 9) and skin rash (16% exon 20). 14% discontinued treatment due to toxicity. The PFS with alpelisib is lower than reported in the SOLAR1 study and is similar to Bylieve study, whose population is most similar to our sample. Exon 20 carriers are those who obtain a higher PFS, as well as those who receive it with an antiestrogen. Exon 9 carriers have a higher rate of G3 AE With respect to inh-CDK4/6, the PFS corresponds to the literature, with exon 20 carriers obtaining a higher PFS.