Immunomodulatory effects of CDK4/6 inhibitors

The dysregulation of the cell cycle is one of the hallmarks of cancer. Cyclin-dependent kinase 4 (CDK4) and CDK6 play crucial roles in regulating cell cycle and other cellular functions. CDK4/6 inhibitors have achieved great success in treating breast cancers and are currently being tested extensively in other tumor types as well. Accumulating evidence suggests that CDK4/6 inhibitors exert antitumor effects through immunomodulation aside from cell cycle arrest. Here we outline the immunomodulatory activities of CDK4/6 inhibitors, discuss the immune mechanisms of drug resistance and explore avenues to harness their immunotherapeutic potential when combined with immune checkpoint inhibitors (ICIs) or chimeric antigen receptor (CAR) T-cell therapy to improve the clinical outcomes.     

Genome wide CRISPR/Cas9 screen identifies the coagulation factor IX (F9) as a regulator of senescence

During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.Subject terms: Senescence, Tumour biomarkers     

Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27

The use of CDK4/6 inhibitors in the treatment of a wide range of cancers is an area of ongoing investigation. Despite their increasing clinical use, there is limited understanding of the determinants of sensitivity and resistance to these drugs. Recent data have cast doubt on how CDK4/6 inhibitors arrest proliferation, provoking renewed interest in the role(s) of CDK4/6 in driving cell proliferation. As the use of CDK4/6 inhibitors in cancer therapies becomes more prominent, an understanding of their effect on the cell cycle becomes more urgent. Here, we investigate the mechanism of action of CDK4/6 inhibitors in promoting cell cycle arrest. Two main models explain how CDK4/6 inhibitors cause G1 cell cycle arrest, which differ in their dependence on the CDK inhibitor proteins p21 and p27. We have used live and fixed single-cell quantitative imaging, with inducible degradation systems, to address the roles of p21 and p27 in the mechanism of action of CDK4/6 inhibitors. We find that CDK4/6 inhibitors can initiate and maintain a cell cycle arrest without p21 or p27. This work clarifies our current understanding of the mechanism of action of CDK4/6 inhibitors and has implications for cancer treatment and patient stratification.     

Targeting the cell cycle in breast cancer: towards the next phase

Deregulation of the cell cycle is a hallmark of cancer that enables limitless cell division. To support this malignant phenotype, cells acquire molecular alterations that abrogate or bypass control mechanisms in signaling pathways and cellular checkpoints that normally function to prevent genomic instability and uncontrolled cell proliferation. Consequently, therapeutic targeting of the cell cycle has long been viewed as a promising anti-cancer strategy. Until recently, attempts to target the cell cycle for cancer therapy using selective inhibitors have proven unsuccessful due to intolerable toxicities and a lack of target specificity. However, improvements in our understanding of malignant cell-specific vulnerabilities has revealed a therapeutic window for preferential targeting of the cell cycle in cancer cells, and has led to the development of agents now in the clinic. In this review, we discuss the latest generation of cell cycle targeting anti-cancer agents for breast cancer, including approved CDK4/6 inhibitors, and investigational TTK and PLK4 inhibitors that are currently in clinical trials. In recognition of the emerging population of ER+ breast cancers with acquired resistance to CDK4/6 inhibitors we suggest new therapeutic avenues to treat these patients. We also offer our perspective on the direction of future research to address the problem of drug resistance, and discuss the mechanistic insights required for the successful implementation of these strategies.     

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.     

CDK4/6信号通路靶向治疗剂在癌症治疗中的研究进展

多数癌症的发生发展都具有细胞周期高度活化的特性。细胞周期蛋白依赖性激酶4/6 (CDK4/6)不仅在细胞有丝分裂中发挥了巨大作用,而且参与了衰老、凋亡和组蛋白调节等诸多生物学过程,并在多种癌症的发生发展中被异常激活。FDA批准了Palbociclib、Ribociclib和Abemaciclib等3种靶向CDK4/6的抑制剂,在临床上也取得了显著的疗效,有效地延长了内分泌治疗耐药的乳腺癌患者以及其他多种类型癌症患者的生存期。但这些抑制剂的临床应用也面临着获得性耐药等问题。文中综述了CDK4/6参与的生物调控过程,及其抑制剂在癌症治疗中的应用和面临的耐药性挑战。     

Vorinostat-induced autophagy switches from a death-promoting to a cytoprotective signal to drive acquired resistance

Histone deacetylase inhibitors (HDACi) have shown promising activity against hematological malignancies in clinical trials and have led to the approval of vorinostat for the treatment of cutaneous T-cell lymphoma. However, de novo or acquired resistance to HDACi therapy is inevitable, and their molecular mechanisms are still unclear. To gain insight into HDACi resistance, we developed vorinostat-resistant clones from the hematological cell lines U937 and SUDHL6. Although cross-resistant to some but not all HDACi, the resistant cell lines exhibit dramatically increased sensitivity toward chloroquine, an inhibitor of autophagy. Consistent with this, resistant cells growing in vorinostat show increased autophagy. Inhibition of autophagy in vorinostat-resistant U937 cells by knockdown of Beclin-1 or Lamp-2 (lysosome-associated membrane protein 2) restores sensitivity to vorinostat. Interestingly, autophagy is also activated in parental U937 cells by de novo treatment with vorinostat. However, in contrast to the resistant cells, inhibition of autophagy decreases sensitivity to vorinostat. These results indicate that autophagy can switch from a proapoptotic signal to a prosurvival function driving acquired resistance. Moreover, inducers of autophagy (such as mammalian target of rapamycin inhibitors) synergize with vorinostat to induce cell death in parental cells, whereas the resistant cells remain insensitive. These data highlight the complexity of the design of combination strategies using modulators of autophagy and HDACi for the treatment of hematological malignancies.     

A systematic review and meta-analysis of immune checkpoint therapy in relapsed or refractory non-Hodgkin lymphoma; a friend or foe?

Over the last decades, a revolution has occurred in oncology with the development of immune checkpoint inhibitors (ICIs). Following tremendous successes in solid tumors, interest has risen to explore these inhibitors in hematologic malignancies; while Hodgkin's lymphoma (HL) has shown overwhelming achievements, available data on different types of non-Hodgkin's lymphoma (NHL) vary considerably. To the best of our knowledge, no meta-analysis has assessed the efficacy and safety of ICI therapy in relapsed or refractory NHL patients. Meta-analysis of the included studies (n = 29) indicated PD-1 may probably be the more attractive ICI target rather than PD-L1 and CTLA-4 in NHL patients. Also, there is a plausible correlation between NHL subtypes and response to ICI therapy. While MF, ENKTL, RT, and PMBCL showed promising responses to ICI monotherapy, neither FL nor DLBCL had satisfactory responses; further necessitating novel strategies such as the application of ICIs in combination with other treatment strategies. Notably, among different combinations, BTK inhibitors showed an obvious improvement as compared to ICI monotherapy in both FL and DLBCL, however, the best results were obtained when ICI was combined with anti-CD20 monoclonal antibodies. Finally, while most NHL patients who received ICI treatment have experienced mild AEs, larger trials with long-term follow-up are required to confirm the safety, as well as the efficacy, of ICI therapy in NHL patients.     

Efficacy of a combination therapy targeting CDK4/6 and autophagy in a mouse xenograft model of t(8;21) acute myeloid leukemia

One of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML) is t(8;21). Although patients with t(8;21) AML have a more favorable prognosis than other cytogenetic subgroups, relapse is still common and novel therapeutic approaches are needed. A recent study showed that t(8;21) AML is characterized by CCND2 deregulation and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells. In this study, we examined the in vivo effects of co-inhibiting CDK4/6 and autophagy. We used a mouse model in which t(8;21)-positive Kasumi-1 cells were subcutaneously inoculated into NOD/Shi-scid IL2Rg^null mice. The mice were treated with the autophagy inhibitor chloroquine (CQ), a CDK4/6 inhibitor (either abemaciclib or palbociclib), or a CDK4/6 inhibitor plus CQ. After 20 days of treatment, tumor volume was measured, and immunostaining and transmission electron microscopy observations were performed. There was no change in tumor growth in CQ-treated mice. However, mice treated with a CDK4/6 inhibitor plus CQ had significantly less tumor growth than mice treated with a CDK4/6 inhibitor alone. CDK4/6 inhibitor treatment increased the formation of autophagosomes. The number of single-strand DNA-positive (apoptotic) cells was significantly higher in the tumors of mice treated with a CDK4/6 inhibitor plus CQ than in mice treated with either CQ or a CDK4/6 inhibitor. These results show that CDK4/6 inhibition induces autophagy, and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells in vivo. The results suggest that inhibiting CDK4/6 and autophagy could be a novel and promising therapeutic strategy in t(8;21) AML.     

Delayed Treatment with Systemic (S)-Roscovitine Provides Neuroprotection and Inhibits In Vivo CDK5 Activity Increase in Animal Stroke Models

Background

Although quite challenging, neuroprotective therapies in ischemic stroke remain an interesting strategy to counter mechanisms of ischemic injury and reduce brain tissue damage. Among potential neuroprotective drug, cyclin-dependent kinases (CDK) inhibitors represent interesting therapeutic candidates. Increasing evidence indisputably links cell cycle CDKs and CDK5 to the pathogenesis of stroke. Although recent studies have demonstrated promising neuroprotective efficacies of pharmacological CDK inhibitors in related animal models, none of them were however clinically relevant to human treatment.

Methodology/Principal Findings

In the present study, we report that systemic delivery of (S)-roscovitine, a well known inhibitor of mitotic CDKs and CDK5, was neuroprotective in a dose-dependent manner in two models of focal ischemia, as recommended by STAIR guidelines. We show that (S)-roscovitine was able to cross the blood brain barrier. (S)-roscovitine significant in vivo positive effect remained when the compound was systemically administered 2 hrs after the insult. Moreover, we validate one of (S)-roscovitine in vivo target after ischemia. Cerebral increase of CDK5/p25 activity was observed 3 hrs after the insult and prevented by systemic (S)-roscovitine administration. Our results show therefore that roscovitine protects in vivo neurons possibly through CDK5 dependent mechanisms.

Conclusions/Significance

Altogether, our data bring new evidences for the further development of pharmacological CDK inhibitors in stroke therapy.     

Enhanced dermatologic toxicity following concurrent treatment with palbociclib and radiation therapy: A case report

BackgroundCyclin-dependent kinase (CDK) 4/6 inhibitors represent a new class of targeted therapy options for the treatment of estrogen receptor-positive (ER+) human epidermal growth factor 2-negative (HER2-) metastatic breast cancer. There are currently no published prospective data on the safety of use of radiation treatment with palbociclib.CaseWe describe the case of a patient with metastatic breast cancer who received radiation treatment to a metastatic supraclavicular lymph node to planned 60 Gy in 30 fractions while on palbociclib, a selective inhibitor of CDK4/6. The patient developed early radiation toxicities including esophagitis and dermatitis that progressed to a severe left neck skin breakdown in the radiation field, resulting in the need for hospitalization. She had a break in treatment but was able to finish the radiation without palbociclib. Her tumor responded well to the treatment and her side effects healed.DiscussionTo our knowledge this is the first case to report on concurrent palbociclib and radiation use, with resultant enhanced radiation effects that required hospitalization for symptom management. Several preclinical studies have shown synergistic effects of radiation and both in vivo and in vitro experiments resulting in improved survival and decreased cell proliferation, respectively, through enhanced G1 cell cycle arrest.ConclusionThis case highlights the importance of using caution when combining radiation with the new targeted therapies. Until more data becomes available, physicians are recommended to exercise clinical judgment when deciding on whether to continue or discontinue a CDK4/6 inhibitor in a patient who may need radiation.     

Discovery of novel 9H-purin derivatives as dual inhibitors of HDAC1 and CDK2

HDAC and CDK inhibitors have been demonstrated to be synergistically in suppressing cancer cell proliferation and inducing apoptosis. In this work, we incorporated the pharmacophore groups of HDACs and CDKs inhibitors into one molecule to design and synthesize a series of purin derivatives as HDAC/CDK dual inhibitors. The lead compound 6d, showing good HDAC1 and CDK2 inhibitory activity with IC₅₀ values of 5.8 and 56 nM, respectively, exhibited attractive potency against several cancer cell lines in vitro. This work may lead to the discovery of a novel scaffold and potential dual HDAC/CDK inhibitors.     

Insight into the Interactions between Novel Isoquinolin-1,3-Dione Derivatives and Cyclin-Dependent Kinase 4 Combining QSAR and Molecular Docking

Several small-molecule CDK inhibitors have been identified, but none have been approved for clinical use in the past few years. A new series of 4-[(3-hydroxybenzylamino)-methylene]-4H-isoquinoline-1,3-diones were reported as highly potent and selective CDK4 inhibitors. In order to find more potent CDK4 inhibitors, the interactions between these novel isoquinoline-1,3-diones and cyclin-dependent kinase 4 was explored via in silico methodologies such as 3D-QSAR and docking on eighty-one compounds displaying potent selective activities against cyclin-dependent kinase 4. Internal and external cross-validation techniques were investigated as well as region focusing, bootstraping and leave-group-out. A training set of 66 compounds gave the satisfactory CoMFA model (q ² = 0.695, r ² = 0.947) and CoMSIA model (q ² = 0.641, r ² = 0.933). The remaining 15 compounds as a test set also gave good external predictive abilities with r ² _pred values of 0.875 and 0.769 for CoMFA and CoMSIA, respectively. The 3D-QSAR models generated here predicted that all five parameters are important for activity toward CDK4. Surflex-dock results, coincident with CoMFA/CoMSIA contour maps, gave the path for binding mode exploration between the inhibitors and CDK4 protein. Based on the QSAR and docking models, twenty new potent molecules have been designed and predicted better than the most active compound 12 in the literatures. The QSAR, docking and interactions analysis expand the structure-activity relationships of constrained isoquinoline-1,3-diones and contribute towards the development of more active CDK4 subtype-selective inhibitors.     

Targeting epigenetic mechanisms to overcome venetoclax resistance

The BH-3 mimetic venetoclax overcomes apoptosis and therapy resistance caused by high expression of BCL2 or loss of BH3-only protein function. Although a promising therapy for hematologic malignancies, increased expression of anti-apoptotic MCL-1 or BCL-XL, as well as other resistance mechanisms prevent a durable response to venetoclax. Recent studies demonstrate that agents targeting epigenetic mechanisms such as DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, histone methyltransferase EZH2 inhibitors, or bromodomain reader protein inhibitors may disable oncogenic gene expression signatures responsible for venetoclax resistance. Combination therapies including venetoclax and epigenetic therapies are effective in preclinical models and the subject of many current clinical trials. Here we review epigenetic strategies to overcome venetoclax resistance mechanisms in hematologic malignancies.     

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.     

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.