Chemotherapy and CDK4/6 Inhibitors: Unexpected Bedfellows Patrick J

Published OnlineFirst June 16, 2020; DOI: 10.1158/1535-7163.MCT-18-1161

MOLECULAR CANCER THERAPEUTICS | REVIEW

Chemotherapy and CDK4/6 Inhibitors: Unexpected Bedfellows Patrick J. Roberts1, Vishnu Kumarasamy2, Agnieszka K. Witkiewicz2,3, and Erik S. Knudsen2,4

ABSTRACT ◥ Cyclin-dependent kinases 4 and 6 (CDK4/6) have emerged as ation between CDK4/6i and chemotherapy. Furthermore, the com- important therapeutic targets. Pharmacologic inhibitors of these bination of CDK4/6i and chemotherapy is being tested in clinical kinases function to inhibit cell-cycle progression and exert other trials to both enhance antitumor efﬁcacy and limit toxicity. Exploi- important effects on the tumor and host environment. Because of tation of the noncanonical effects of CDK4/6i could also provide an their impact on the cell cycle, CDK4/6 inhibitors (CDK4/6i) have impetus for future studies in combination with chemotherapy. been hypothesized to antagonize the antitumor effects of cytotoxic Thus, while seemingly mutually exclusive mechanisms are at play, chemotherapy in tumors that are CDK4/6 dependent. However, the combination of CDK4/6 inhibition and chemotherapy could there are multiple preclinical studies that illustrate potent cooper- exemplify rational medicine.

CDK4/6 in Cell-cycle Progression phosphorylate many substrates, CDK4/6 has a very limited repertoire of targets (18). CDK4 and CDK6 selectively phosphorylate the RB Cyclin dependent kinases (CDK) are serine/threonine kinases that tumor suppressor protein and additional members of the RB regulate the sequential progression of the cell cycle in eukaryotic family (18–21). RB-family proteins function as transcriptional cor- organisms. The molecular functions of these kinases in different epressors and limit the expression of E2F target genes that include phases of the cell cycle have been well characterized (1, 2). The multiple genes required for cell-cycle progression, DNA replication, cell-cycle machinery in higher eukaryotes is tightly regulated by the and mitotic progression (22, 23). The phosphorylation of RB, which is presence of more than 10 proteins in the CDK family that can have initiated by CDK4 or CDK6 serves to limit transcriptional repression overlapping and distinct functions (2). Cell-cycle initiation occurs in and enable progression through latter phases of the cell cycle defining G phase, which is conventionally governed by the activation of CDK4 1 the canonical CDK4/6-RB pathway (Fig. 1A). and CDK6 kinases that are downstream of mitogenic signals (3–5). The requirement for CDK4/6 in cell division has been interrogated The catalytic activity of CDK4 and CDK6 is positively regulated by the utilizing multiple approaches and has illustrated important features of binding of D-type cyclins (D1, D2, and D3). Expression of D-type the cell cycle. The inhibition of CDK4/6 by the expression of endog- cyclins is induced in response to mitogenic stimuli and remains high as enous inhibitors (e.g., p16INK4A) potently arrests cells that contain a the cells progress to the G –S phase boundary (6). Therefore, unlike 1 functional RB protein and subsequently limits gene expression con- other cyclins and CDKs that are regulated by other components of the trolled by RB/E2F (Fig. 1A). Multiple experimental methods (e.g., cell-cycle machinery, the expression of D-type cyclins, and by exten- antibody injection, RNAi, etc.) have further suggested that D-type sion, CDK4/6-associated kinase activity, largely depend on mitogenic cyclins and/or CDK4/6 activity are generally important for progres- signaling pathways (7, 8). Transcription of D-type cyclins is intimately sion from G1/S in normal cells as well as multiple cancer models (24). linked to multiple pathways that coalesce to lead to the accumulation of These findings contrast with studies in mouse models that clearly transcripts (7, 9, 10). Mitogenic signaling pathways also regulate the demonstrate that the cell cycle can proceed with genetic deletion of stability and localization of these proteins (11, 12). Importantly, a host CDK4 and 6 or deletion of all D-type cyclins (25, 26). In this context, of growth-inhibitory mechanisms also impact CDK4/6 activity, adaptation occurs in many tissues by enabling CDK2 or CDK1 activity including the induction of endogenous CDK4/6-specific inhibitors to drive cell cycle entry. However, genetic suppression of CDK4/6 with specific stresses (e.g., CDKN2A which encodes p16INK4A), and activity can limit or block tumor development in select models (27–30). active mechanisms of cyclin D1 degradation (13, 14). Thus, CDK4/6 This was clearly shown in the context of HER2-driven breast cancer activity acts as a sensor linking multiple signaling pathways to the where CDK4/6 activity is required both for tumor etiology and initiation of the cell cycle (15–17). maintenance (31). CDK4/6 regulates the cell cycle through phosphorylation of key substrates. Unlike the prototypical CDK1 and CDK2, which can Pharmacologic Inhibitors of CDK4/6— Mechanisms of Action and Resistance 1 2 G1 Therapeutics, Research Triangle Park, North Carolina. Center for Person- Because of the function of CDK4/6 in coordinating cell division, alized Medicine, Roswell Park Cancer Institute, Buffalo, New York. 3Department 4 pharmacologic inhibitors have been developed as anticancer drugs. of Pathology, Roswell Park Cancer Institute, Buffalo, New York. Department of fi Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York. There are ve selective CDK4/6 inhibitors (CDK4/6i); palbociclib (PD0332991), ribociclib (LEE011), abemaciclib (LY2835219), trilaci- Corresponding Author: Erik S. Knudsen, Roswell Park Cancer Center, Elm and clib (G1T28), and lerociclib (G1T38; refs. 32–38). Currently, three of Carlton Streets, Buffalo, NY 14263. Phone: 716-845-1224; Fax: 716-845-5908; þ E-mail: [email protected] these drugs are FDA-approved for the treatment of ER metastatic breast cancer based on multiple randomized clinical trials (palbociclib, Mol Cancer Ther 2020;19:1575–88 ribociclib, abemaciclib). While all of these compounds are selective doi: 10.1158/1535-7163.MCT-18-1161 for CDK4/6, palbociclib, ribociclib, abemaciclib, and lerociclib are Ó2020 American Association for Cancer Research. formulated for oral long-term dosing. Trilaciclib was formulated

AACRJournals.org | 1575

Roberts et al.

A Co-repressors Cyc D RB1 CDK4/6 E2F

P P RB1

S-phase E2F Mitosis DNA-repair

B Cyc D Co-repressors CDK4/6-inhibitor RB1 CDK4/6

E2F G1-arrest

C Deregulated oncogenic pathways

Co-repressors CDK4/6-inhibitor 2 Cyc E

1 CDK2 Cyc D

CDK4/6 E2F P P RB1

S-phase E2F Mitosis Cytokinesis

Figure 1. Different cell cycle states in cancer. A, The Canonical G1/S regulatory circuit: CDK4/6 kinase activity is stimulated downstream of mitogenic/oncogenic signals to initiate the phosphorylation of RB and related proteins. Phosphorylation facilitates the derepression of E2F-family of transcription factors that drive the expression of many genes required for DNA replication, mitosis, and cell division. B, CDK4/6 dependent cells: in cells, tumors, or tissues that are dependent on CDK4/6 activity treatment with pharmacological inhibitors yields the robust activation of RB. This event limits other CDK activities and represses the expression of essential genes for

cellular division resulting in a G1–G0–like arrest. C, CDK4/6-independent cells: there are clearly two distinct states that yield CDK4/6 independent proliferation. (1) Loss of RB as occurs in a subset of human tumors removes the down-stream target and as such inhibition of CDK4/6 has minimal efﬁcacy in controlling cell cycle. (2) Through various mechanisms RB phosphorylation can remain during pharmacologic inhibition. This cell-cycle plasticity can be generated through either CDK4/6 or CDK2 complexes and is prevalent in a number of tumor types that retain the RB tumor suppressor.

speciﬁcally for intravenous delivery and short half-life with the activation (Fig. 1B) and suppress the expression of genes that are intended goal of preventing chemotherapy-induced host toxicities. conventionally regulated by the E2F family of transcription fac- Consistent with their mechanism of action, all CDK4/6i have cytostatic tors (39, 40). Because many of these genes are involved in core – activity that is associated with RB-dependent suppression of the G1 S functions of DNA replication and mitotic progression, and are con- transition (32, 36). Pharmacologic CDK4/6i mimic the effect of RB sidered essential for proliferation, the magnitude of transcriptional

1576 Mol Cancer Ther; 19(8) August 2020 MOLECULAR CANCER THERAPEUTICS

Chemotherapy and CDK4/6 Inhibitors: Unexpected Bedfellows

repression downstream from CDK4/6 inhibition is critical for cyto- prevent chemotherapy-induced cellular damage of normal cells that static activity. harbor an intact RB pathway (refs. 54, 55; Fig. 2A). One of the common Multiple determinants of response to CDK4/6 inhibition are being side effects of chemotherapy is myelosuppression, that can lead to the elucidated through both preclinical investigation and the analysis of exhaustion of hematopoietic stem and progenitor cells (HSPCs; clinical specimens (Fig. 1C). This work has illustrated that there are refs. 56–58). multiple cell-cycle–related alterations present in models or tumors Trilaciclib (G1T28) has been developed to specifically prevent (e.g., RB loss or overexpression of cyclin E) that are associated with chemotherapy-induced myelosuppression (38). Trilaciclib maintains resistance to CDK4/6 inhibitors (refs. 41–44; Fig. 1C). Conversely, a selective and reversible G1 arrest in the RB-proficient HPSCs and a number of oncogenic signaling pathways (e.g., RAS/MAPK, prevents or mitigates the acute and long-term hematopoietic toxicity PTEN/PI3K, or HIPPO) have emerged as contributing to of the cytotoxic chemotherapeutic agent, 5-fluorouracil, when admin- resistance (45–47). These derangements enable escape from istered concurrently (38, 55). Similarly, the cytostatic effect of CDK4/6i CDK4/6 inhibition by facilitating the inactivation/phosphorylation can prevent or mitigate the hematopoietic toxicity of ionizing radiation of RB even in the presence of the pharmacologic CDK4/6i. This is by preventing HSPCs from immediately entering the cell cycle when believed to occur due to “plasticity,” which is associated with either the cells sense the radiation-induced DNA damage (54). incomplete inhibition of CDK4/6 or the ability of CDK2 to initiate the To translate these findings to the clinic, three randomized, placebo- phosphorylation of RB. While inhibition of CDK4/6 can arrest cells, controlled, double-blind clinical trials designed to evaluate the mye- dual inhibition of CDK4 and CDK2 has been shown to be required for lopreservation effects of trilaciclib versus placebo in combination with durable responses in preclinical models (48). In addition, the hyper- chemotherapy have been completed in small-cell lung carcinoma activation of CDK2 kinase in breast cancer cells due to overexpression (SCLC; refs. 59–62; Table 1). SCLC was chosen as the first clinical of cyclin E1/cyclin E2 drives resistance to CDK4/6i (49). Conversely, setting to test the myelopreservation benefit of trilaciclib because: RNAi-mediated knockdown of CCNE1 or CDK2 along with CDK4/6 (i) standard-of-care chemotherapy regimens are myelosuppressive, inhibition can reverse acquired resistance to CDK4/6 inhibition in (ii) SCLC replicates independently of CDK4/6 due to obligate loss of select models (50–52). Thus, the level of CDK2 activity during RB (63), thereby minimizing theoretical concerns related to chemo- response is an important determinant and potential biomarker for therapy antagonism, and (iii) SCLC treated in the first-line setting is a the efficacy of CDK4/6i. chemosensitive tumor, which provided an optimal background upon which to demonstrate that trilaciclib does not antagonize chemotherapy efficacy. In all three studies, trilaciclib demonstrated consistent The Spectrum of CDK4/6i Tumor clinical benefit across myelosuppression endpoints, including highly Sensitivities and Theoretical statistically significant improvements for both primary endpoints: the Intersection with Chemotherapy duration of severe neutropenia in cycle 1 (a surrogate for febrile neutropenia and infections) and the percentage of patients with severe From preclinical and clinical studies, emerging data indicate that neutropenia. Furthermore, integrated analysis from the three studies there are tumors that are sensitive to CDK4/6i, but that many of these demonstrated statistically significant improvement across multiple sensitive tumors develop adaptive resistance mechanisms. In these hematopoietic lineages; including neutrophils (duration of severe contexts, CDK4/6i combination therapies can enhance the efficacy and neutropenia in cycle 1, percentage of patients with severe neutropenia), durability of the tumor response. In contrast, there are a subset of red blood cells (RBC; percentage of patients with grade 3 or 4 anemia, cancers that are intrinsically CDK4/6-independent (e.g., as a conse- percentage of patients receiving RBC transfusions on or after 5 weeks, quence of RB loss; refs. 33, 53). The consequences of the addition of the rate of RBC transfusions on or after 5 weeks), and platelets CDK4/6i to chemotherapy must be considered within this framework. (percentage of patients with grade 3 or 4 thrombocytopenia; ref. 59). In patients with CDK4/6-independent tumors, the anticipated clinical Consistent with the improvement in chemotherapy safety, patient- benefit would be to protect normal cells from chemotherapy as the reported outcome (PRO) measures demonstrated an improved expe- normal cells are sensitive to CDK4/6 inhibition and the tumor cells are rience for patients receiving trilaciclib, including improved measures insensitive to CDK4/6 inhibition (Fig. 2A). In patients with CDK4/6- of fatigue (59). Importantly, the addition of trilaciclib to chemotherapy dependent tumors, there may be opportunities to enhance antitumor did not have an adverse effect on antitumor efficacy (60–62). efficacy; however, there is a theoretical risk that CDK4/6 inhibition in Similar to SCLC, triple-negative breast cancer (TNBC) is thought to this setting may antagonize the intended cytotoxicity of the chemo- be a mostly CDK4/6-independent tumor, based on both tumor therapy (Fig. 2B). Preclinical and clinical data suggest that the risk of genetics and the relatively poor response of such tumors to CDK4/6i chemotherapy antagonism by CDK4/6i is not as well understood as therapy in preclinical studies (64, 65). Trilaciclib has been tested in initially thought. In addition, the molecular determinants of CDK4/6 combination with gemcitabine and carboplatin in patients with met- independence and dependence are complex, such that it can be difficult astatic TNBC (66). In this study, the addition of trilaciclib to chemo- to identify those tumors that truly rely upon CDK4/6 for proliferation. therapy generally did not improve myelosuppression endpoints; how- We discuss these approaches using the “theoretical” binary tumor ever, there were positive trends for RBC and platelet measures, and classification of CDK4/6-independent and -dependent and acknowl- patients in the trilaciclib arms received significantly more chemother- edge that the spectrum of dependence may actually be continuous. apy than the control. In contrast, the antitumor efficacy results demonstrated a clinically meaningful survival benefit in both combi- CDK4/6-Independent Tumors—Host nation groups compared with the chemotherapy alone control group. Median progression-free survival was 5.7 months (95% CI: 3.4–9.2) for Protection the control group compared with 9.4 (6.1–13.0; HR: 0.60, P ¼ 0.13) – P ¼ The use of CDK4/6i to arrest cells in the G1 phase in patients with and 7.3 (6.2 12.19; HR: 0.59, 0.12) for the two trilaciclib cancer who are being treated with chemotherapy may not seem to be groups. Median overall survival was 12.6 months (6.3–15.6) for the intuitive. However, this biological phenomenon can be exploited to chemotherapy control group compared with 20.1 (10.2–not reached;

AACRJournals.org Mol Cancer Ther; 19(8) August 2020 1577

Roberts et al.

A CDK4/6-inhibitor Slow cell division Mitigate toxicity

CDK4/6-independent tumor CDK4/6-dependent host tissues -RB-deﬁcient (e.g. small cell lung cancer) -Hematopoietic stem cells -Refractory (e.g. triple negative breast cancer) -Kidney -Hair follicle -Salivary function

B Chemotherapy

Apoptosis Replication mediated DNA damage Irreparable DNA-damage Rapidly CDK4/6 inhibitor dividing Checkpoint tumor Deﬁcits cells Mitotic catastrophe Aberrant mitotic entry

Irreparable Chromosome-damage

Figure 2. Differential response to CDK4/6 inhibition determines mode of interaction with chemotherapy. A, CDK4/6 independent tumors: in CDK4/6-independent tumor models, by definition the pharmacologic inhibitors will have minimal effect. SCLC tumors exhibit near universal loss of the RB tumor suppressor, while triple-negative breast cancer exhibits multiple mechanisms that render CDK4/6 independence. In contrast, the host-tissues will be responsive to the CDK4/6 inhibitor and the reduced cell division could be expected to mitigate the toxicity associated with chemotherapy in specific tissue settings. B, Conceptual frame-work for antagonism: chemotherapy represents a diverse class of drugs that exploit the rapid division of tumor cells that impinges on DNA replication or mitotic division. This sensitivity is often enhanced as a result of cell-cycle checkpoint deficits in tumor cells. Ultimately, apoptosis, necrosis, or mitotic catastrophe lead to cytotoxic activity. Because CDK4/6 inhibitors can slow cell-cycle progression they could negatively impact on the efficacy of chemotherapy.

HR: 0.33, P ¼ 0.028) and 17.8 (12.9–not reached; HR: 0.34, P ¼ 0.0023) described the antagonistic effects of combining a CDK4/6i with for the two trilaciclib þ chemotherapy groups. While patients receiv- chemotherapy (refs. 50, 68–70; Table 2). In breast cancer cell lines, ing trilaciclib received more chemotherapy, it is unlikely that this can xenografts, and GEMM models, treatment with CDK4/6i can limit the explain the magnitude of survival benefit achieved with transient acute induction of tumor-specific toxicity with taxanes, anthracyclines, CDK4/6 inhibition. Instead, an alternative mechanism of action and platinum-agents (68, 70–72). These effects are RB-dependent and related to enhanced antitumor immunity is more likely as discussed link the antagonism of chemotherapy cytotoxicity with the cell-cycle in more detail below. pause induced by CDK4/6 inhibition (68, 70, 71). In considering these data, it is important to appreciate that most studies utilized conditions where CDK4/6 inhibition elicits profound cell-cycle inhibition and Antagonism of Chemotherapy- that effects measured on antagonism were relatively short term; mediated Cytotoxicity in CDK4/6- however, it should not be discounted that such antagonism could be dependent Preclinical Models clinically relevant and caution should be taken to evaluate whether these effects will be seen in specific clinical settings. On the basis of the intracellular targets of many chemotherapeutic agents, it is evident that dividing cells are more chemosensitive than arrested cells, which underlies the therapeutic index of such Potential Cooperation between agents (67). Considering the mechanism of action of CDK4/6i in CDK4/6i and Chemotherapy in Cancer inhibiting cell division through activation of the RB pathway, it is hypothesized that concurrent CDK4/6 inhibition may antagonize the Therapy in Preclinical Models cytotoxic effects of chemotherapeutic agents in tumors that are CDK4/ In contrast to the above reports, a number of preclinical studies 6-dependent (Fig. 2B). Indeed, a number of preclinical reports have suggest that the combination of chemotherapy and CDK4/6 inhibition

1578 Mol Cancer Ther; 19(8) August 2020 MOLECULAR CANCER THERAPEUTICS

Chemotherapy and CDK4/6 Inhibitors: Unexpected Bedfellows

Table 1. Clinical studies of CDK4/6 inhibitors with chemotherapy.

CDK4/6 Study title inhibitor Chemotherapy Phase Description NCT#

Phase II study of carboplatin, Trilaciclib Carboplatin, Etoposide, 2 This is a study to investigate the NCT03041311 etoposide, and atezolizumab with and Atezolizumab potential clinical benefit of trilaciclib or without trilaciclib in patients (G1T28) in preserving the bone with untreated extensive stage marrow and the immune system, small-cell lung cancer and enhancing antitumor efficacy when administered with carboplatin, etoposide, and atezolizumab (E/P/A) therapy in first-line treatment for patients with newly diagnosed extensive-stage SCLC. Phase II study of the safety, efficacy, Trilaciclib Gemcitabine and 2 This is a study to investigate the NCT02978716 and pharmacokinetics of G1T28 in Carboplatin potential clinical benefit of trilaciclib patients with metastatic triple- (G1T28) in preserving the bone negative breast cancer receiving marrow and the immune system, gemcitabine and carboplatin and enhancing chemotherapy chemotherapy antitumor efficacy when administered prior to carboplatin and gemcitabine (GC therapy) for patients with metastatic triple- negative breast cancer. Phase Ib/IIa safety and Trilaciclib Topotecan 1/2 This is a study to investigate the NCT02514447 pharmacokinetic study of G1T28 potential clinical benefit of trilaciclib in patients with previously (G1T28) in preserving the bone treated extensive stage small-cell marrow and the immune system, lung cancer (SCLC) receiving and enhancing chemotherapy topotecan chemotherapy antitumor efficacy when administered prior to topotecan in patients previously treated for extensive-stage SCLC. Phase Ib/IIa safety and Trilaciclib Carboplatin and 1/2 This is a study to investigate the NCT02499770 pharmacokinetic study of G1T28 Etoposide potential clinical benefit of trilaciclib in patients with extensive stage (G1T28) in preserving the bone small cell lung cancer (SCLC) marrow and the immune system, receiving etoposide and and enhancing chemotherapy carboplatin antitumor efficacy when administered prior to carboplatin and etoposide in first line treatment for patients with newly diagnosed extensive-stage SCLC. A phase I study of palbociclib, a CDK Palbociclib Cytarabine, 1 AINV18P1 is a phase I study where NCT03792256 4/6 inhibitor, in combination with Methotrexate, palbociclib is administrated in chemotherapy in children with Hydrocortisone, combination with a standard re- relapsed acute lymphoblastic Doxorubicin, induction platform in pediatric leukemia (ALL) or lymphoblastic Prednisolone, relapsed Acute Lymphoblastic lymphoma (LL) Vincristine, Leukemia (ALL) and lymphoblastic Pegaspargase, lymphoma (LL). Prednisone A phase I study of the CDK4/6 Palbociclib 5-Fluorouracil and 1 The purpose of this study is to test the NCT01522989 inhibitor PD-0332991, Oxaliplatin safety and effectiveness of a new 5-fluorouracil, and oxaliplatin in combination of drugs, palbociclib, patients with advanced solid 5-fluorouracil and oxaliplatin for tumor malignancies patients with advanced solid tumor malignancies. Phase IB study of PD-0332991 in Palbociclib T-DM1 1 This is a phase IB inter-patient dose NCT01976169 combination with T-DM1 in the escalation study of PD-0332991 in treatment of patients with combination with T–DM1 in patients advanced HER2 (human with recurrent or metastatic HER2- epidermal growth factor receptor positive breast cancer after prior 2)-positive breast cancer trastuzumab or other HER2- directed therapies. (Continued on the following page)

AACRJournals.org Mol Cancer Ther; 19(8) August 2020 1579

Roberts et al.

Table 1. Clinical studies of CDK4/6 inhibitors with chemotherapy. (Cont'd ) CDK4/6 Study title inhibitor Chemotherapy Phase Description NCT#

A phase I trial of PD0332991 and Palbociclib Paclitaxel 1 This study is a phase I, single arm, NCT01320592 paclitaxel in patients with Rb- open-label trial of PD0332991 in expressing advanced breast combination with paclitaxel in cancer patients with Rb-expressing metastatic breast cancer. An open-label phase IB study of Palbociclib Nab-Paclitaxel 1 ThisisaPhase1,openlabel, NCT02501902 palbociclib (oral CDK4/6 multi center, multiple dose, inhibitor) plus abraxane (nab- dose escalation, safety, paclitaxel) in patients with pharmacokinetic and metastatic pancreatic ductal pharmacodynamic study of adenocarcinoma palbociclib in combination with nab-P, in sequential cohorts of adult patients with mPDAC, with MTD expansion cohort(s). A Phase 1 study of palbociclib in Palbociclib Carboplatin or 1 This phase I trial studies the side NCT02897375 combination with cisplatin or Cisplatin effects and best dose of carboplatin in advanced solid palbociclib with cisplatin or malignancies carboplatin in treating patients with solid tumors that have spread to other places and usually cannot be cured or controlled with treatment. A phase 1b/2 study of the oral Ribociclib Docetaxel and 1/2 This is a phase Ib/II open label clinical NCT02494921 CDK4/6 inhibitor LEE011 Prednisone trial in patients with metastatic (ribociclib) in combination with castration resistant prostate cancer. docetaxel plus prednisone in The objective of the phase Ib metastatic castration-resistant portion of the study is to establish prostate cancer the maximum tolerated dose (MTD) and dose-limiting toxicities (DLT) of docetaxel (75 mg/m2, i.v. q21 days) and prednisone (5 mg orally, BID) in combination with ribociclib in escalating oral daily doses in patients with metastatic CRPC with prior resistance to abiraterone and/ or enzalutamide who have not undergone prior chemotherapy for metastatic disease. A phase I study of CDK4/6 inhibitor Ribociclib Gemcitabine 1 This phase I trial studies the side NCT02414724 LEE011 combined with effects and best dose of ribociclib gemcitabine in patients with and gemcitabine hydrochloride in advanced solid tumors or treating patients with solid tumors lymphoma or lymphoma that have spread to other places in the body and usually cannot be cured or controlled with treatment Phase I study of CDK4/6 inhibitor Ribociclib Gemcitabine 1 This phase I trial studies ribociclib and NCT03237390 ribociclib (LEE011) combined with gemcitabine hydrochloride in gemcitabine in patients with treating patients with solid tumors advanced solid tumors that have spread to other places in the body. Study of ribociclib with everolimus Ribociclib Everolimus and 1/2 The purpose of this study is NCT02732119 þ exemestane in HRþ HER2- Exemestane determine if the triplet locally advanced/metastatic combination of ribociclib, breast cancer postprogression on everolimus and exemastane is CDK 4/6 Inhibitor. (TRINITI-1) effective in the treatment of locally advanced/metastatic breast cancer following treatment with a CDK 4/6 inhibitor (Continued on the following page)

1580 Mol Cancer Ther; 19(8) August 2020 MOLECULAR CANCER THERAPEUTICS

Chemotherapy and CDK4/6 Inhibitors: Unexpected Bedfellows

Table 1. Clinical studies of CDK4/6 inhibitors with chemotherapy. (Cont'd ) CDK4/6 Study title inhibitor Chemotherapy Phase Description NCT#

Phase Ib trial of LEE011 With Ribociclib Everolimus and 1 This study evealuates the safety and NCT01857193 everolimus (RAD001) and Exemestane tolerability of the triplet exemestane in the treatment of combination of LEE011 þ hormone receptor positive HER2 everolimus þ exemestane in negative advanced breast cancer patients na€ve or refractory to CDK4/6 inhibitor-based therapy, and the safety and tolerability of the doublet combination of LEE011 þ exemestane in patients refractory to CDK4/6 inhibitor-based therapy. An open-label, phase Ib/II clinical Ribociclib T-DM1, 1/2 This study tests the combination of NCT02657343 trial of Cdk 4/6 inhibitor, ribociclib Trastuzumab, ribociclib in combination with (LEE011), in combination with Fulvestrant T-DM1, trastuzumab, or trastuzumab or T-Dm1 for trastuzumab plus fulvestrant in advanced/metastatic Her2- patients with advanced/metastatic positive breast cancer Her2þ breast cancer. A phase I trial of ribocilcib (LEE011) Ribociclib Paclitaxel 1 This is a phase I study to assess the NCT02599363 and weekly paclitaxel in patients safety and MTD of paclitaxel þ with Rbþ advanced breast cancer ribociclib (LEE011) in patients with Rbþ, advanced breast cancer. Dose escalation will be performed using standard 3 þ 3 dosing strategy. A phase Ib/II study of LEE011 and Ribociclib TACE 2 The purpose of this study is determine NCT02524119 chemoembolization in patients whether the combination therapy with advanced hepatocellular with LEE011 and chemoembolization carcinoma in patients with locally advanced hepatocellular carcinoma not amenable to curative therapies will provide greater efficacy than chemoembolization alone with a tolerable safety profile. A phase Ib/II study of the oral Ribociclib Docetaxel 1/2 This is an open-label study of ribociclib NCT02494921 CDK4/6 inhibitor LEE011 Prednisone (dosed at the RP2D) in combination (ribociclib) in combination with with docetaxel and prednisone to docetaxel plus prednisone in determine the efficacy and safety of metastatic castration-resistant the treatment combination in prostate cancer patients with metastatic castration resistant prostate cancer. A phase Ib study of LY2835219 in Abemaciclib Pemetrexed 1 The main purpose of this study is to NCT02079636 combination with multiple single- Gemcitabine evaluate the safety and tolerability agent options for patients with of abemaciclib in combination with stage IV NSCLC another anti-cancer drug in participants with NSCLC that is advanced or has spread to other parts of the body (stage IV). A phase II study of abemaciclib in Abemaciclib Pemetrexed 2 The main purpose of this study is to NCT02308020 patients with brain metastases Gemcitabine evaluate the safety and effectiveness secondary to hormone receptor of the study drug known as positive breast cancer, non-small- abemaciclib in participants with cell lung cancer, or melanoma hormone receptor positive breast cancer, non-small cell lung cancer (NSCLC), or melanoma that has spread to the brain. Some cohorts allow concurrent pemetrexed and/or gemcitabine. A phase II trial program exploring SHR6390 Capecitabine 2 Patients previously failing NCT04095390 the integration of novel HER2- transtuzumab therapy are targeted tyrosine kinase inhibitor randomized to pyrotinib and pyrotinib and CDK4/6 inhibitor SHR6390 plus capcitabine, SHR6390 into current letrozole, or placebo. chemotherapy/endocrine therapy regimes for prior trastuzumab-treated advanced HER2-positive breast cancer

AACRJournals.org Mol Cancer Ther; 19(8) August 2020 1581

Roberts et al.

Table 2. Preclinical studies demonstrating some evidence of antagonism between CDK4/6 inhibitors and chemotherapy.

Author Tumor type Context Chemo Outcome

Franco et al. Human PDA In vitro Gemcitabine * Observed antagonism when palbociclib was added to gemcitabine but Oncotarget 2014 5FU cooperation between palbociclib and 5FU. * Palbociclib reduced TS, the target of 5-FU. * Differential cell cycle sensitivity to palbociclib despite uniform suppression of RB phosphorylation.

McClendon et al. Human TNBC In vitro Doxorubicin * In RB-proﬁcient TNBC cell lines, co-treatment yielded cooperative cytostatic Cell Cycle 2012 In vivo effects but reduced doxorubicin mediated cytotoxicity.

Dean et al. Human TNBC In vitro Doxorubicin * Mostly a biochemical demonstration that CDK4/6 inhibition blocks cells in G1 and JBC 2012 In vivo Paclitaxel is associated with reduced E2F gene expression even in the presence of Radiation chemotherapy. * Pre-treatment and continuous palbociclib reduced paclitaxel effects in outgrowth assays, but pre and concurrent synchronization experiments enhanced activity in vivo. * Showed CDK4/6 inhibition pushes DNA repair from HR to NHEJ and palbociclib leads to reduction of Ku70 needed for NHEJ, which implies that CDK4/6 inhibition may impair a cancer cells ability to repair chemotherapy induced DNA damage.

Roberts et al. TNBC In vitro Carboplatin * Protection of “normal” cell line and bone marrow from chemotherapy. JCI 2012 HER2 In vivo Doxorubicin * Concurrent palbociclib did not antagonize carboplatin in TNBC model. Etoposide * Concurrent palbociclib plus carboplatin treatment led to tumor regression in Camptothecin Her2 model, but depth of response was not as deep as single agent carboplatin. Paclitaxel

Konecny et al. Human ovarian In vitro Carboplatin * Concurrent palbociclib treatment with carboplatin or paclitaxel resulted in Clin Ca Re. 2011 synergism, however pre-treatment resulted in antagonism.

Cretella et al. Human TNBC In vitro Paclitaxel * Pre-treatment with palbociclib sensitized cells to paclitaxel (sequential) and Scientiﬁc there was no difference in efﬁcacy when paclitaxel was added 0, 4, 8 hours after Reports 2019 palbociclib. * Simultaneous treatment showed an antagonistic effect.

can have cooperative antitumor effects; similar observations are demonstrated a role for MDR (multidrug resistance; P-glycoprotein) beginning to emerge from clinical studies. Palbociclib, ribociclib, and in paclitaxel resistance, an effect counteracted by both CDK4 siRNA abemaciclib have been shown to enhance, rather than antagonize, and palbociclib treatment (76). Finally, multiple studies have dem- chemotherapy cytotoxicity when combined with camptothecin, car- onstrated that CDK4/6 inhibition can enhance chemotherapy-induced boplatin, cisplatin, docetaxel, doxorubicin, 5-FU, gemcitabine, irino- apoptosis (76, 79, 85), and that CDK4/6 are upstream regulators of tecan, paclitaxel, and temozolomide (36, 73–85). These effects were transcription factors that control global gene expression leading to shown in RB-proficient in vitro and in vivo models of non–small cell changes in metabolism, DNA repair, and cell plasticity, all of which lung carcinoma (NSCLC), ovarian cancer, gastric cancer, TNBC, can render a cancer cell more susceptible to chemotherapy cytotox- atypical teratoid rhabdoid tumors, Ewing sarcoma, pancreatic cancer, icity (88). Collectively, these results suggest that the net effect of and glioblastoma using both sequential and concurrent dosing sche- concomitant CDK4/6 inhibition during chemotherapy exposure in dules (refs. 36, 73–85; Table 3). patients with CDK4/6-dependent tumors will provide cooperation While the CDK4/6-RB-E2F axis is responsible for controlling rather than antagonism (Table 3). expression of genes required for cell-cycle progression, DNA replication, and mitotic progression (23, 39), the unexpected observations of Strategies for Incorporating CDK4/6i cooperation described above may be due to other less well understood mechanisms. One mechanistic explanation of the enhanced, rather into Chemotherapy Regimens than antagonistic activity of combination CDK4/6i plus chemotherapy On the basis of the available preclinical and clinical data, there are regimens, has been the reduced expression of specific E2F-regulated several therapeutic strategies by which CDK4/6i can be incorporated genes, whose products are targeted by chemotherapy (Fig. 3A). Pal- into standard chemotherapy regimens to provide therapeutic benefitto bociclib treatment reduces thymidylate synthase (TS; 5FU target), patients: Topoisomerase 1, and Topoisomerase 2 alpha expression. These effects on gene expression could potentially enhance the response to select Protection of normal tissues chemotherapies by limiting the threshold needed for efficacy of As described above, protection of HSPCs to reduce dose-limiting chemotherapy (70, 77, 86). Similarly, E2F regulates the expression of myelosuppression has been demonstrated preclinically and multiple genes required for DNA damage repair and thus would limit clinically (38, 60–62, 68, 89). While myelosuppression is recognized as the ability of tumor cells to recover from chemotherapy-mediated a common complication of chemotherapy, damage to other normal damage. Consistent with the impact on DNA repair machinery, it has tissues including the gastrointestinal track, kidney, and hair follicles also been shown that CDK4/6i can cooperate with PARP inhibitors occurs. CDK4/6 inhibition has been shown to ameliorate kidney injury ostensibly by limiting the ability of damaged cells to carry out HR- in preclinical models following both cisplatin treatment and acute renal mediated repair (ref. 87; Fig. 3A). Conversely, Gao and colleagues ischemia, and to provide intestinal radioprotection (39, 90, 91). In

1582 Mol Cancer Ther; 19(8) August 2020 MOLECULAR CANCER THERAPEUTICS

Chemotherapy and CDK4/6 Inhibitors: Unexpected Bedfellows

Table 3. Preclinical studies demonstrating enhanced efﬁcacy with CDK4/6 inhibitors þ chemotherapy treatment.

Author Inhibitor Tumor type Context Chemotherapy Outcome

Hamilton et al. Palbociclib SCLC In vitro Camptothecins * Palbociclib reduced TOPO1 expression and enhanced Molecules 2014 camptothecin activity

Gelbert et al. Inv New Abemaciclib Lung cancer In vivo Gemcitabine * Abemaciclib plus gemcitabine sequentially or Drug 2014 concurrently was better than either agent alone.

Hashizume et al. Palbociclib ATRT and In vitro Radiation * Palbociclib treatment reduced phospho-RB levels and Neuro-Oncology glioblastoma In vivo pre-, concurrent and post-palbociclib treatment 2016 enhanced gH2AX formation, enhanced tumor response and improved overall survival.

O'Brien et al. MCT 2018 Abemaciclib TNBC In vitro Docetaxel, Carboplatin * No antagonism seen in either phospho-RB high/p16 In vivo low (MDA-231) or phospho-RB low/p16 high (HCC70) TNBC mouse models when abemaciclib was combined with docetaxel (concurrent treatment). * Additionally, in MDA-231 cells, abemaciclib enhanced activity with concurrent treatment for 5 days with either docetaxel or carboplatin.

Iyengar et al. Ribociclib Ovarian Ca In vitro Cisplatin * Ribociclib þ cisplatin improved efﬁcacy in vitro and Oncotarget 2018 In vivo in vivo compared to cisplatin alone in RB1 competent, CDK4/6 sensitive ovarian models.

Dowless et al. Clin Abemaciclib Ewing sarcoma In vitro Doxorubicin, Etoposide, * Abemaciclib increased IC50 of various Ca Re 2018 In vivo Cisplatin, Paclitaxel, chemotherapies (doxorubicin, etoposide, cisplatin, Temozolomide paclitaxel) in vitro, but enhanced anti-tumor efﬁcacy Irinotecan of doxorubicin and temozolomide/irinotecan in vivo.

Chou et al. Gut 2018 Palbociclib PDAC In vitro Gemcitabine * Palbociclib improved efﬁcacy of gemcitabine and In vivo Paclitaxel * Gemcitabine/paclitaxel in RB-high patient derived PDAC models. * Improved endpoints include synergy, apoptosis, reduced metastasis (in vitro and in vivo), primary and recurrent (2nd-line therapy) tumor growth.

Raub et al. Drug Metab Abemaciclib Glioblastoma In vivo Temozolomide * Abemaciclib improved anti-tumor efﬁcacy and Dispos 2015 survival when added to temozolomide in U87MG xenograft model and rat orthotopic model.

Wang et al. Int J Mol Palbociclib Gastric Ca In vitro 5FU * Palbociclib produced a dose dependent G1 arrest in Med. 2018 gastric cancer cells and enhanced 5FU mediated cytotoxicity. * Proteomic analysis demonstrated that palbociclib altered expression of proteins involved in the regulation of cell death, cell cycle, cell growth, proliferation, and cell migration.

Gao et al. Cell Oncol. Palbociclib Ovarian In vitro Paclitaxel * Inhibition of CDK4(and 6) by palbociclib or CDK4- 2017 speciﬁc siRNA increased the sensitivity of both RB- positive and -negative ovarian cancer cells to paclitaxel. * Combination treatment enhanced apoptosis as measured by increased PARP cleavage and reduced Bcl-xL and Survivin. * Demonstrated a role for MDR in paclitaxel resistance and showed that palbociclib and CDK4 siRNA may act in part to reduce MDR activity.

Zhang et al. Cancer CINK4 NSCLC In Vitro Paclitaxel * CDK4 siRNA signiﬁcantly increased paclitaxel Biology & Therapy. sensitivity in KRAS mutation-positive H23 cells. 2013 * CINK4 demonstrated concentration- and time- dependent anti-proliferative activity in 5 NSCLC cell lines. * Combined CINK4 and paclitaxel produced synergistic anti-proliferative activity and increased apoptosis through reduced cyclin D1 and Bcl-2 in KRAS mutation-positive cancer cells.

Cao et al. Oncogene Palbociclib Squam lung In vitro Paclitaxel * Palbociclib enhanced in vitro and in vivo efﬁcacy 2019 In vivo through RB-dependent E2F mediated alterations in senescence, G2–M spindle checkpoint, and angiogenesis. * Note: this series of experiments used sequential treatment schedule. In vivo Pac D1 Palbo D2–6for 4 weekly cycles (Continued on the following page)

AACRJournals.org Mol Cancer Ther; 19(8) August 2020 1583

Roberts et al.

Table 3. Preclinical studies demonstrating enhanced efﬁcacy with CDK4/6 inhibitors þ chemotherapy treatment. (Cont'd ) Author Inhibitor Tumor type Context Chemotherapy Outcome

Kumarasamy et al. Palbociclib, PDAC In vitro Gemcitabine * CDK4/6i prevented the outgrowth of tumor cells Oncogene 2019 Ribociclib In vivo Docetaxel following the release from gemcitabine by inhibiting the re-entry of cells to cell-cycle through the downregulation of DNA replication and repair genes. * CDK4/6i in combination with docetaxel resulted in a cooperative inhibitory effect on cell-cycle progression by inhibiting the CDK2 kinase activity

Salvador-Barbero Palbociclib PDAC In vitro Paclitaxel * Sequential administration of palbociclib following et al. Cancer cell In vivo taxol treatment cooperatively prevented cell 2020 proliferation in PDAC cells and PDX models. * Palbociclib-mediated repression of proteins involved in homologous recombination prevented the ability of cells to recover from chromosomal damage.induced by chemotherapy

Abbreviations: ATRT, atypical teratoid rhabdoid tumor; Ca, cancer; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer; Squam lung, squamous lung cancer; TNBC, triple negative breast cancer; PDAC, Pancreatic ductal adenocarcinoma.

addition, alopecia, while not life threatening, is one of the most distres- ovarian cancer models, following the release from cisplatin-mediated sing side effects of chemotherapy. Similar to other tissues, transient S-phase arrest, tumor cells undergo normal cell-cycle progression and CDK4/6 inhibition has been shown to protect hair follicles from taxane- proliferation, which is significantly blocked by CDK4/6 inhibition, induced damage in preclinical models (92). While clinical benefit for this indicating a positive interaction (80). Similarly, the targeted micro- approach has been shown in a CDK4/6-independent setting (SCLC), the tubule poison trastuzumab-emtansine (T-DM1) displayed a cooper- question remains whether it can be employed in CDK4/6-dependent ative antitumor effect where CDK4/6i could block the recovery of tumors without antagonizing chemotherapy antitumor efficacy. In vivo residual cells following T-DM1 treatment (99). Presently, there are a evaluation of trilaciclib with chemotherapy in CDK4/6-sensitive breast number of clinical trials that explore the interaction of CDK4/6 cancer models has not shown antagonism at the tumor level, and with inhibition and chemotherapy using dosing strategies to enhance some models, the combination shows enhanced antitumor efficacy (93). durability of response (Table 1). In one of the first reported trials, Interestingly, subset analysis of patients in the trilaciclib metastatic the combination of paclitaxel with palbociclib appeared to have TNBC (mTNBC) study using PAM50 and other molecular stratification efficacy in heavily pretreated breast cancer (100). approaches revealed no antagonism, and demonstrated improved PFS As CDK4/6i are not associated with cumulative toxicity, which is a and OS across all groups (55, 66, 94, 95). common feature of chemotherapy, they can be given for long periods of time. Therefore, chronic administration of a CDK4/6i in the Concurrent interactions, maintenance therapy, and staggered maintenance treatment setting, after the tumor has been debulked strategies to enhance antitumor efficacy and chemotherapy discontinued could lead to improved patient out- Because the mechanisms of CDK4/6i and chemotherapy action comes by delaying tumor progression and or allowing the host are distinct, there could be drug interactions that would enhance the immune system to eliminate the residual disease (80). efficacy of each class of agent. Chemotherapy is well known to impact CDK biology at multiple points that would be expected to Enhancing the response to immunotherapy enhance the cytostatic response to CDK4/6 inhibition (73). For There is significant preclinical data demonstrating that CDK4/6i example, chemotherapy can impact CDK2 activity via the induction can enhance immune checkpoint inhibitor (ICI) efficacy through of the endogenous CDK inhibitor p21 or loss of the CDC25a protein enhanced T-cell activation, increased antigen presentation, phosphatase that would yield increased inhibitory phosphorylation increased expression of PD-L1, and reduced T-cell exclusion and on CDK2 (Fig. 3B). This cooperation has been illustrated in models immune evasion gene signature (88, 101, 102) In the clinic, of pancreatic cancer and other tumor models that do not show chemotherapy has successfully been used to enhance ICI robust response to CDK4/6i (96, 97). Regarding resistance to efficacy (103–108) through induction of immunogenic cell death, chemotherapy, as discussed above, CDK4/6i can impact the expres- enhancement of immunosurveillance, and T-cell activity, and sion of genes associated with DNA repair and dNTP metabolism. reduction of immunosuppressive cell types (109–112). Despite These effects could be broadly relevant to chemotherapy that these benefits, chemotherapy-induced myelosuppression and induces DNA damage or is associated with nucleotide metabolism immunosuppression may limit the full benefitofcombinatorial or function (Fig. 3A). Whether these interactions manifest clinically treatments with ICIs. Given that intratumor immune cells are remains unclear, although multiple clinical trials are interrogating highly proliferative, one strategy to further enhance chemothera- CDK4/6i and chemotherapy combinations where there is clear py/ICI combinations is through transient CDK4/6 inhibition dur- overlap in the treatment. ing chemotherapy exposure. Trilaciclib has been shown to favor- Given the canonical action of chemotherapy and CDK4/6i, it is ably alter the tumor immune microenvironment through transient appealing to separate/stagger the dosing of the chemotherapy and the T-cell inhibition (113, 114). Treatment of immunocompetent CDK4/6i. In this context, the chemotherapy can have the desired tumor models with trilaciclib plus chemotherapy/ICI combina- impact of killing the tumor cells, while the CDK4/6i prevents the tions significantly improved antitumor efficacy and survival com- expansion of cells that are not killed by the chemotherapy (98). In pared with chemotherapy/ICI combinations (113, 114).

1584 Mol Cancer Ther; 19(8) August 2020 MOLECULAR CANCER THERAPEUTICS

Chemotherapy and CDK4/6 Inhibitors: Unexpected Bedfellows

A Base/Nucleotide excision repair or Mismatch repair LIG1, MSH2, NEIL3, NUDT1, POLE, PCNA, UNG, DDB2, FANCD2, POLE, RPA3

Non-homologous end joining Co-repressors FEN1, TRIP13

RB1 Homologous recombination-mediated repair BARD1, BLM, BRCA1, BRCA2, BRIP1, DCLRE1B, DCLRE1A, DNA2, EME1, EXO1, E2F FANCA, FANCB, FANCC, FANC1, FANCD2, FANCE, FANCG, POLA1, POLE, POLD1, POLD3, RAD51, RAD51C, RAD51AP1, RAD54L, RBBP8, RECQL4, RFWD3, RMI1, RMI2, RPA3, TOPB1, UBE2T, USP1, XRCC2, XRCC3

DNA damage response CHEK1, H2AFX, PARP2, RAD9B, RAD18, SMC1A, TIMELESS, TP73, TOPBP1, WDHD1

dNTP metabolism RRM2, DTYMK, DUT; TYMS, MTHFD1; HPRT1; PRTFDC1; RRM1; DHFR

B Chemotherapy

CDC25A p21

Cyc E

CDK2 Cyc D CDK4/6-inhibitor CDK4/6 Co-repressors

RB1

E2F Cell-cycle arrest

Figure 3. Cellular response to CDK4/6 inhibition in combination with chemotherapy. A, Downstream from the activation of the RB pathway are the repression of multiple genes involved in different pathways that could impinge on different features of response to distinct chemotherapeutic agents. B, There are multiple mechanisms through which chemotherapy impinges on the activity of CDK2 and CDK4/6 complexes that would be expected to enhance the efﬁcacy of pharmacologic CDK4/6 inhibitors. For example, CDC25A is rapidly degraded or the CDK2-inhibitor P21CIP1 is rapidly induced as a speciﬁc response to chemotherapy. Conversely, degradation of cyclin D1 and inhibition of CDK4/6 complexes is a consequence of different chemotherapy agents that induce S-phase block.

is still needed. However, identifying “truly” CDK4/6-dependent Summary and Underexplored Areas tumors remains elusive as predictive biomarkers have not been In summary, while it was originally hypothesized that CDK4/6i validated in clinical practice. Identification and validation of such combined with chemotherapy could potentially result in antagonistic biomarkers would allow clinical testing of CDK4/6i þ chemotherapy antitumor effects (at least in CDK4/6-dependent tumors), emerging combinations in a homogeneous CDK4/6-dependent tumor popula- data suggest that these agents can be safely combined using different tion to definitively determine whether CDK4/6 inhibition during clinical strategies to enhance antitumor efficacy and/or reduce che- chemotherapy exposure interferes with the intended antitumor effi- motherapy-induced toxicity. There are a number of unanswered cacy of chemotherapy. In addition, understanding the contribution of questions whose answers could help guide implementation of these antitumor effects arising from cell-cycle inhibition in tumor cells strategies in the clinic. Understanding which of these clinical strategies versus nontumor cells (e.g., cancer associated fibroblasts and immune would be best employed in tumors that are “truly” CDK4/6-dependent cells) and through noncanonical biological processes controlled by

AACRJournals.org Mol Cancer Ther; 19(8) August 2020 1585

Roberts et al.

CDK4/6 will further aid the rational design of novel CDK4/6i plus Acknowledgments chemotherapy and/or immunotherapy regimens. The authors acknowledge their colleagues at Roswell Park and G1 Therapeutics for thoughtful discussion and review of the manuscript. This work was supported by Disclosure of Potential Conflicts of Interest grants AKW and ESK from the NIH CA211878 and CA247362. P.J. Roberts is a senior director of translational medicine at G1 Therapeutics and has ownership interest (including patents) in G1 Therapeutics. No potential conflicts Received February 26, 2020; revised April 17, 2020; accepted June 10, 2020; of interest were disclosed by the other authors. published first June 16, 2020.

References 1. Beach D, Durkacz B, Nurse P. Functionally homologous cell cycle control genes 25. Malumbres M, Sotillo R, Santamaria D, Galan J, Cerezo A, Ortega S, et al. in budding and fission yeast. Nature 1982;300:706–9. Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and 2. Nurse P, Thuriaux P. Regulatory genes controlling mitosis in the fission yeast Cdk6. Cell 2004;118:493–504. Schizosaccharomyces pombe. Genetics 1980;96:627–37. 26. Kozar K, Ciemerych MA, Rebel VI, Shigematsu H, Zagozdzon A, Sicinska E, 3. Baker SJ, Reddy EP. CDK4: a key player in the cell cycle, development, and et al. Mouse development and cell proliferation in the absence of D-cyclins. cancer. Genes Cancer 2012;3:658–69. Cell 2004;118:477–91. 4. Duronio RJ, Xiong Y. Signaling pathways that control cell proliferation. 27. Santamaria D, Barriere C, Cerqueira A, Hunt S, Tardy C, Newton K, et al. Cdk1 Cold Spring Harb Perspect Biol 2013;5:a008904. is sufficient to drive the mammalian cell cycle. Nature 2007;448:811–5. 5. Morrison DK. MAP kinase pathways. Cold Spring Harb Perspect Biol 2012;4: 28. Malumbres M, Barbacid M. Is cyclin D1-CDK4 kinase a bona fide cancer target? a011254. Cancer Cell 2006;9:2–4. 6. Pagano M, Theodoras AM, Tam SW, Draetta GF. Cyclin D1-mediated inhi- 29. Yu Q, Geng Y, Sicinski P. Specific protection against breast cancers by cyclin D1 bition of repair and replicative DNA synthesis in human fibroblasts. Genes Dev ablation. Nature 2001;411:1017–21. 1994;8:1627–39. 30. Landis MW, Pawlyk BS, Li T, Sicinski P, Hinds PW. Cyclin D1-dependent 7. Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, et al. kinase activity in murine development and mammary tumorigenesis. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter Cancer Cell 2006;9:13–22. through distinguishable regions. J Biol Chem 1995;270:23589–97. 31. Yu Q, Sicinska E, Geng Y, Ahnstrom M, Zagozdzon A, Kong Y, et al. 8. Sherr CJ. D-type cyclins. Trends Biochem Sci 1995;20:187–90. Requirement for CDK4 kinase function in breast cancer. Cancer Cell 2006; 9. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimulating factor 1 9:23–32. regulates novel cyclins during the G1 phase of the cell cycle. Cell 1991;65: 32. Fry DW, Harvey PJ, Keller PR, Elliott WL, Meade M, Trachet E, et al. Specific 701–13. inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated 10. Brown JR, Nigh E, Lee RJ, Ye H, Thompson MA, Saudou F, et al. Fos family antitumor activity in human tumor xenografts. Mol Cancer Ther 2004;3: members induce cell cycle entry by activating cyclin D1. Mol Cell Biol 1998;18: 1427–38. 5609–19. 33. Finn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai AJ, et al. PD 0332991, 11. Diehl JA. Cycling to cancer with cyclin D1. Cancer Biol Ther 2002;1:226–31. a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of 12. Diehl JA, Zindy F, Sherr CJ. Inhibition of cyclin D1 phosphorylation on luminal estrogen receptor-positive human breast cancer cell lines in vitro. threonine-286 prevents its rapid degradation via the ubiquitin-proteasome Breast Cancer Res 2009;11:R77. pathway. Genes Dev 1997;11:957–72. 34. Tripathy D, Bardia A, Sellers WR. Ribociclib (LEE011): mechanism of action 13. Agami R, Bernards R. Distinct initiation and maintenance mechanisms coop- and clinical impact of this selective cyclin-dependent kinase 4/6 inhibitor in erate to induce G1 cell cycle arrest in response to DNA damage. Cell 2000;102: various solid tumors. Clin Cancer Res 2017;23:3251–62. 55–66. 35. Corona SP, Generali D. Abemaciclib: a CDK4/6 inhibitor for the treatment of 14. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control HRþ/HER2 advanced breast cancer. Drug Des Devel Ther 2018;12:321–30. causing specific inhibition of cyclin D/CDK4. Nature 1993;366:704–7. 36. Gelbert LM, Cai S, Lin X, Sanchez-Martinez C, Del Prado M, Lallena MJ, et al. 15. Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M, Pestell R, et al. Preclinical characterization of the CDK4/6 inhibitor LY2835219: in-vivo cell The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl cycle-dependent/independent anti-tumor activities alone/in combination with Acad Sci U S A 1999;96:5522–7. gemcitabine. Invest New Drugs 2014;32:825–37. 16. Joyce D, Bouzahzah B, Fu M, Albanese C, D'Amico M, Steer J, et al. Integration 37. Bisi JE, Sorrentino JA, Jordan JL, Darr DD, Roberts PJ, Tavares FX, et al. of Rac-dependent regulation of cyclin D1 transcription through a nuclear Preclinical development of G1T38: a novel, potent and selective inhibitor of factor-kappaB-dependent pathway. J Biol Chem 1999;274:25245–9. cyclin dependent kinases 4/6 for use as an oral antineoplastic in patients with 17. Alt JR, Cleveland JL, Hannink M, Diehl JA. Phosphorylation-dependent CDK4/6 sensitive tumors. Oncotarget 2017;8:42343–58. regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular 38. Bisi JE, Sorrentino JA, Roberts PJ, Tavares FX, Strum JC. Preclinical charac- transformation. Genes Dev 2000;14:3102–14. terization of G1T28: a novel CDK4/6 inhibitor for reduction of chemotherapy- 18. Matsushime H, Ewen ME, Strom DK, Kato JY, Hanks SK, Roussel MF, et al. induced myelosuppression. Mol Cancer Ther 2016;15:783–93. Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) 39. DiRocco DP, Bisi J, Roberts P, Strum J, Wong KK, Sharpless N, et al. CDK4/6 for mammalian D type G1 cyclins. Cell 1992;71:323–34. inhibition induces epithelial cell cycle arrest and ameliorates acute kidney 19. Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ. Direct binding of cyclin injury. Am J Physiol Renal Physiol 2014;306:F379–88. D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the 40. Knudsen ES, Witkiewicz AK. Defining the transcriptional and biological cyclin D-dependent kinase CDK4. Genes Dev 1993;7:331–42. response to CDK4/6 inhibition in relation to ERþ/HER2 breast cancer. 20. Meyerson M, Harlow E. Identification of G1 kinase activity for cdk6, a novel Oncotarget 2016;7:69111–23. cyclin D partner. Mol Cell Biol 1994;14:2077–86. 41. Dean JL, Thangavel C, McClendon AK, Reed CA, Knudsen ES. Therapeutic 21. Farkas T, Hansen K, Holm K, Lukas J, Bartek J. Distinct phosphorylation events CDK4/6 inhibition in breast cancer: key mechanisms of response and failure. regulate p130- and p107-mediated repression of E2F-4. J Biol Chem 2002;277: Oncogene 2010;29:4018–32. 26741–52. 42. Konecny GE, Winterhoff B, Kolarova T, Qi J, Manivong K, Dering J, et al. 22. Ren B, Cam H, Takahashi Y, Volkert T, Terragni J, Young RA, et al. E2F Expression of p16 and retinoblastoma determines response to CDK4/6 inhi- integrates cell cycle progression with DNA repair, replication, and G(2)/M bition in ovarian cancer. Clin Cancer Res 2011;17:1591–602. checkpoints. Genes Dev 2002;16:245–56. 43. Wiedemeyer WR, Dunn IF, Quayle SN, Zhang J, Chheda MG, Dunn GP, et al. 23. Burkhart DL, Sage J. Cellular mechanisms of tumour suppression by the Pattern of retinoblastoma pathway inactivation dictates response to CDK4/6 retinoblastoma gene. Nat Rev Cancer 2008;8:671–82. inhibition in GBM. Proc Natl Acad Sci U S A 2010;107:11501–6. 24. Knudsen ES, Witkiewicz AK. The strange case of CDK4/6 inhibitors: mechan- 44. Marzec M, Kasprzycka M, Lai R, Gladden AB, Wlodarski P, Tomczak E, et al. isms, resistance, and combination strategies. Trends Cancer 2017;3:39–55. Mantle cell lymphoma cells express predominantly cyclin D1a isoform and are

1586 Mol Cancer Ther; 19(8) August 2020 MOLECULAR CANCER THERAPEUTICS

Chemotherapy and CDK4/6 Inhibitors: Unexpected Bedfellows

highly sensitive to selective inhibition of CDK4 kinase activity. Blood 2006;108: metastatic triple-negative breast cancer: a multicentre, randomised, open-label, 1744–50. phase 2 trial. Lancet Oncol 2019;20:1587–601. 45. de Leeuw R, McNair C, Schiewer MJ, Neupane NP, Brand LJ, Augello MA, et al. 67. Gudkov AV, Komarova EA. Radioprotection: smart games with death. J Clin MAPK reliance via acquired CDK4/6 inhibitor resistance in cancer. Invest 2010;120:2270–3. Clin Cancer Res 2018;24:4201–14. 68. Roberts PJ, Bisi JE, Strum JC, Combest AJ, Darr DB, Usary JE, et al. Multiple 46. Cornell L, Wander SA, Visal T, Wagle N, Shapiro GI. MicroRNA-mediated roles of cyclin-dependent kinase 4/6 inhibitors in cancer therapy. J Natl Cancer suppression of the TGF-beta pathway confers transmissible and reversible Inst 2012;104:476–87. CDK4/6 inhibitor resistance. Cell Rep 2019;26:2667–80. 69. McClendon AK, Dean JL, Rivadeneira DB, Yu JE, Reed CA, Gao E, et al. 47. Formisano L, Lu Y, Servetto A, Hanker AB, Jansen VM, Bauer JA, et al. Aberrant CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline ther- FGFR signaling mediates resistance to CDK4/6 inhibitors in ERþ breast cancer. apy. Cell Cycle 2012;11:2747–55. Nat Commun 2019;10:1373. 70. Dean JL, McClendon AK, Knudsen ES. Modification of the DNA damage 48. Patel P, Tsiperson V, Gottesman SRS, Somma J, Blain SW. Dual inhibition of response by therapeutic CDK4/6 inhibition. J Biol Chem 2012;287:29075–87. CDK4 and CDK2 via targeting p27 tyrosine phosphorylation induces a potent 71. Witkiewicz AK, Chung S, Brough R, Vail P, Franco J, Lord CJ, et al. Targeting and durable response in breast cancer cells. Mol Cancer Res 2018;16:361–77. the vulnerability of RB tumor suppressor loss in triple-negative breast cancer. 49. Portman N, Alexandrou S, Carson E, Wang S, Lim E, Caldon CE. Overcoming Cell Rep 2018;22:1185–99. CDK4/6 inhibitor resistance in ER-positive breast cancer. Endocr Relat Cancer 72. Jin D, Tran N, Thomas N, Tran DD. Combining CDK4/6 inhibitors ribociclib 2019;26:R15–R30. and palbociclib with cytotoxic agents does not enhance cytotoxicity. PLoS One 50. Franco J, Witkiewicz AK, Knudsen ES. CDK4/6 inhibitors have potent activity 2019;14:e0223555. in combination with pathway selective therapeutic agents in models of pan- 73. Cao J, Zhu Z, Wang H, Nichols TC, Lui GYL, Deng S, et al. Combining CDK4/6 creatic cancer. Oncotarget 2014;5:6512–25. inhibition with taxanes enhances anti-tumor efficacy by sustained impairment 51. Knudsen ES, Kumarasamy V, Ruiz A, Sivinski J, Chung S, Grant A, et al. Cell of pRB-E2F pathways in squamous cell lung cancer. Oncogene 2019;38: cycle plasticity driven by MTOR signaling: integral resistance to CDK4/6 4125–41. inhibition in patient-derived models of pancreatic cancer. Oncogene 2019; 74. Chou A, Froio D, Nagrial AM, Parkin A, Murphy KJ, Chin VT, et al. Tailored 38:3355–70. first-line and second-line CDK4-targeting treatment combinations in mouse 52. Herrera-Abreu MT, Palafox M, Asghar U, Rivas MA, Cutts RJ, Garcia-Murillas models of pancreatic cancer. Gut 2018;67:2142–55. I, et al. Early adaptation and acquired resistance to CDK4/6 inhibition in 75. Dowless M, Lowery CD, Shackleford T, Renschler M, Stephens J, Flack R, et al. estrogen receptor-positive breast cancer. Cancer Res 2016;76:2301–13. Abemaciclib is active in preclinical models of ewing sarcoma via multipronged 53. Bertucci F, Ng CKY, Patsouris A, Droin N, Piscuoglio S, Carbuccia N, et al. regulation of cell cycle, DNA methylation, and interferon pathway signaling. Genomic characterization of metastatic breast cancers. Nature 2019;569:560–4. Clin Cancer Res 2018;24:6028–39. 54. Johnson SM, Torrice CD, Bell JF, Monahan KB, Jiang Q, Wang Y, et al. 76. Gao Y, Shen J, Choy E, Mankin H, Hornicek F, Duan Z. Inhibition of CDK4 Mitigation of hematologic radiation toxicity in mice through pharmacological sensitizes multidrug resistant ovarian cancer cells to paclitaxel by increasing quiescence induced by CDK4/6 inhibition. J Clin Invest 2010;120:2528–36. apoptosiss. Cell Oncol 2017;40:209–18. 55. Asghar US, Barr AR, Cutts R, Beaney M, Babina I, Sampath D, et al. Single-cell 77. Hamilton G, Klameth L, Rath B, Thalhammer T. Synergism of cyclin- dynamics determines response to CDK4/6 inhibition in triple-negative breast dependent kinase inhibitors with camptothecin derivatives in small cell lung cancer. Clin Cancer Res 2017;23:5561–72. cancer cell lines. Molecules 2014;19:2077–88. 56. Gardner RV, Lerner C, Astle CM, Harrison DE. Assessing permanent damage 78. Hashizume R, Zhang A, Mueller S, Prados MD, Lulla RR, Goldman S, et al. to primitive hematopoietic stem cells after chemotherapy using the competitive Inhibition of DNA damage repair by the CDK4/6 inhibitor palbociclib delays repopulation assay. Cancer Chemother Pharmacol 1993;32:450–4. irradiated intracranial atypical teratoid rhabdoid tumor and glioblastoma 57. Gardner RV. Long term hematopoietic damage after chemotherapy and xenograft regrowth. Neuro Oncol 2016;18:1519–28. cytokine. Front Biosci 1999;4:e47–57. 79. Huang X, Di Liberto M, Jayabalan D, Liang J, Ely S, Bretz J, et al. Prolonged early 58. Mauch P, Constine L, Greenberger J, Knospe W, Sullivan J, Liesveld JL, et al. G(1) arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells Hematopoietic stem cell compartment: acute and late effects of radiation to cytotoxic killing through cell cycle-coupled loss of IRF4. Blood 2012;120: therapy and chemotherapy. Int J Radiat Oncol Biol Phys 1995;31:1319–39. 1095–106. 59. Weiss J SK, Gwaltney C, Daniel D, Adler S, Wo0lfe S, Malik RK, et al. Positive 80. Iyengar M, O'Hayer P, Cole A, Sebastian T, Yang K, Coffman L, et al. CDK4/6 effects of trilaciclib on patient myelosuppression-related symptoms and fucn- inhibition as maintenance and combination therapy for high grade serous tioning: results from three phase 2 randomized, double-blind, placebo- ovarian cancer. Oncotarget 2018;9:15658–72. controlled small cell lung cancer trials. Support Care Cancer 2019;27:S274. 81. O'Brien N, Conklin D, Beckmann R, Luo T, Chau K, Thomas J, et al. Preclinical 60. Weiss JM, Csoszi T, Maglakelidze M, Hoyer RJ, Beck JT, Domine Gomez M, activity of abemaciclib alone or in combination with antimitotic and targeted et al. Myelopreservation with the CDK4/6 inhibitor trilaciclib in patients with therapies in breast cancer. Mol Cancer Ther 2018;17:897–907. small-cell lung cancer receiving first-line chemotherapy: a phase Ib/random- 82. Raub TJ, Wishart GN, Kulanthaivel P, Staton BA, Ajamie RT, Sawada GA, et al. ized phase II trial. Ann Oncol 2019;30:1613–21. Brain exposure of two selective dual CDK4 and CDK6 inhibitors and the 61. Hart LL, Andric ZG, Hussein MA, Ferrarotto R, Beck JT, Subramanian J, et al. antitumor activity of CDK4 and CDK6 inhibition in combination with Effect of trilaciclib, a CDK 4/6 inhibitor, on myelosuppression in patients with temozolomide in an intracranial glioblastoma xenograft. Drug Metab Dispos previously treated extensive-stage small cell lung cancer receiving topotecan. 2015;43:1360–71. J Clin Oncol 2019;37:8505. 83. Wang D, Sun Y, Li W, Ye F, Zhang Y, Guo Y, et al. Antiproliferative effects of the 62. Daniel D, Kuchava V, Bondarenko I, Ivashchuk O, Spigel D, Dasgupta A, et al. CDK6 inhibitor PD0332991 and its effect on signaling networks in gastric 1742PDTrilaciclib (T) decreases myelosuppression in extensive-stage small cell cancer cells. Int J Mol Med 2018;41:2473–84. lung cancer (ES-SCLC) patients receiving first-line chemotherapy plus atezo- 84. Zhang J, Zhou L, Zhao S, Dicker DT, El-Deiry WS. The CDK4/6 inhibitor lizumab. Ann Oncol 2019;30(5 suppl):v713. palbociclib synergizes with irinotecan to promote colorectal cancer cell death 63. George J, Lim JS, Jang SJ, Cun Y, Ozretic L, Kong G, et al. Comprehensive under hypoxia. Cell Cycle 2017;16:1193–200. genomic profiles of small cell lung cancer. Nature 2015;524:47–53. 85. ZhangXH,ChengY,ShinJY,KimJO,OhJE,KangJH.ACDK4/6inhibitor 64. Patnaik A, Rosen LS, Tolaney SM, Tolcher AW, Goldman JW, Gandhi L, et al. enhances cytotoxicity of paclitaxel in lung adenocarcinoma cells harboring Efficacy and safety of abemaciclib, an inhibitor of CDK4 and CDK6, for patients mutant KRAS as well as wild-type KRAS. Cancer Biol Ther 2013;14: with breast cancer, non-small cell lung cancer, and other solid tumors. 597–605. Cancer Discov 2016;6:740–53. 86. Min A, Kim JE, Kim YJ, Lim JM, Kim S, Kim JW, et al. Cyclin E overexpression 65. Witkiewicz AK, Knudsen ES. Retinoblastoma tumor suppressor pathway in confers resistance to the CDK4/6 specific inhibitor palbociclib in gastric cancer breast cancer: prognosis, precision medicine, and therapeutic interventions. cells. Cancer Lett 2018;430:123–32. Breast Cancer Res 2014;16:207. 87. Yi J, Liu C, Tao Z, Wang M, Jia Y, Sang X, et al. MYC status as a determinant of 66. Tan AR, Wright GS, Thummala AR, Danso MA, Popovic L, Pluard TJ, et al. synergistic response to Olaparib and Palbociclib in ovarian cancer. EBioMe- Trilaciclib plus chemotherapy versus chemotherapy alone in patients with dicine 2019;43:225–37.

AACRJournals.org Mol Cancer Ther; 19(8) August 2020 1587

Roberts et al.

88. Klein ME, Kovatcheva M, Davis LE, Tap WD, Koff A. CDK4/6 inhibitors: the 101. Knudsen ES, Pruitt SC, Hershberger PA, Witkiewicz AK, Goodrich DW. Cell mechanism of action may not be as simple as once thought. Cancer Cell 2018; cycle and beyond: exploiting new rb1 controlled mechanisms for cancer 34:9–20. therapy. Trends Cancer 2019;5:308–24. 89. He S, Roberts PJ, Sorrentino JA, Bisi JE, Storrie-White H, Tiessen RG, et al. 102. Deng J, Wang ES, Jenkins RW, Li S, Dries R, Yates K, et al. CDK4/6 inhibition Transient CDK4/6 inhibition protects hematopoietic stem cells from chemo- augments antitumor immunity by enhancing T-cell activation. Cancer Discov therapy-induced exhaustion. Sci Transl Med 2017;9:eaal3986. 2018;8:216–33. 90. Pabla N, Gibson AA, Buege M, Ong SS, Li L, Hu S, et al. Mitigation of acute 103. Gandhi L, Rodriguez-Abreu D, Gadgeel S, Esteban E, Felip E, De Angelis F, et al. kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 Pembrolizumab plus chemotherapy in metastatic non-small-cell lung cancer. functions. Proc Natl Acad Sci U S A 2015;112:5231–6. N Engl J Med 2018;378:2078–92. 91. Wei L, Leibowitz BJ, Wang X, Epperly M, Greenberger J, Zhang L, et al. 104. Horn L, Mansfield AS, Szczesna A, Havel L, Krzakowski M, Hochmair MJ, et al. Inhibition of CDK4/6 protects against radiation-induced intestinal injury in First-line atezolizumab plus chemotherapy in extensive-stage small-cell lung mice. J Clin Invest 2016;126:4076–87. cancer. N Engl J Med 2018;379:2220–9. 92. Purba TS, Ng'andu K, Brunken L, Smart E, Mitchell E, Hassan N, et al. CDK4/6 105. Ott PA, Hodi FS, Kaufman HL, Wigginton JM, Wolchok JD. Combination inhibition mitigates stem cell damage in a novel model for taxane-induced immunotherapy: a road map. J Immunother Cancer 2017;5:16. alopecia. EMBO Mol Med 2019;11:e11031. 106. Paz-Ares L, Luft A, Vicente D, Tafreshi A, Gumus M, Mazieres J, et al. 93. Sorrentino JA BJ, Thompson D, Lai AY, Hall CR, Strum JC, Roberts PJ. Pembrolizumab plus chemotherapy for squamous non-small-cell lung cancer. Trilaciclib, a Cdk4/6 inhibitor, does not impair the efficacy of chemotherapy N Engl J Med 2018;379:2040–51. in Cdk4/6-dependent tumor models. Eur J Cancer 2018;103:e23–e147. 107. Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csoszi T, Fulop A, et al. 94. Lehmann BD, Jovanovic B, Chen X, Estrada MV, Johnson KN, Shyr Y, et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung Refinement of triple-negative breast cancer molecular subtypes: implications cancer. N Engl J Med 2016;375:1823–33. for neoadjuvant chemotherapy selection. PLoS One 2016;11:e0157368. 108. Schmid P, Adams S, Rugo HS, Schneeweiss A, Barrios CH, Iwata H, et al. 95. Prat A, Bianchini G, Thomas M, Belousov A, Cheang MC, Koehler A, et al. Atezolizumab and nab-paclitaxel in advanced triple-negative breast cancer. Research-based PAM50 subtype predictor identifies higher responses and N Engl J Med 2018;379:2108–21. improved survival outcomes in HER2-positive breast cancer in the NOAH 109. North RJ. Cyclophosphamide-facilitated adoptive immunotherapy of an estab- study. Clin Cancer Res 2014;20:511–21. lished tumor depends on elimination of tumor-induced suppressor T cells. 96. Kumarasamy V, Ruiz A, Nambiar R, Witkiewicz AK, Knudsen ES. Chemo- J Exp Med 1982;155:1063–74. therapy impacts on the cellular response to CDK4/6 inhibition: distinct 110. Pfirschke C, Engblom C, Rickelt S, Cortez-Retamozo V, Garris C, Pucci F, et al. mechanisms of interaction and efficacy in models of pancreatic cancer. Immunogenic chemotherapy sensitizes tumors to checkpoint blockade Oncogene 2019;39:1831–45. therapy. Immunity 2016;44:343–54. 97. Salvador-Barbero B, Alvarez-Fernandez M, Zapatero-Solana E, El Bakkali A, 111. Suzuki E, Kapoor V, Jassar AS, Kaiser LR, Albelda SM. Gemcitabine selectively Menendez MDC, Lopez-Casas PP, et al. CDK4/6 inhibitors impair recovery eliminates splenic Gr-1þ/CD11bþ myeloid suppressor cells in tumor-bearing from cytotoxic chemotherapy in pancreatic adenocarcinoma. Cancer Cell 2020; animals and enhances antitumor immune activity. Clin Cancer Res 2005;11: 37:340–53. 6713–21. 98. Cretella D, Fumarola C, Bonelli M, Alfieri R, La Monica S, Digiacomo G, et al. 112. Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G. Immunological aspects of Pre-treatment with the CDK4/6 inhibitor palbociclib improves the efficacy of cancer chemotherapy. Nat Rev Immunol 2008;8:59–73. paclitaxel in TNBC cells. Sci Rep 2019;9:13014. 113. Lai AY, Sorrentino JA, Strum JC, Roberts PJ. Transient exposure of trilaciclib, a 99. Witkiewicz AK, Cox D, Knudsen ES. CDK4/6 inhibition provides a potent CDK4/6 inhibitor, modulates gene expression in tumor immune infiltrates to adjunct to Her2-targeted therapies in preclinical breast cancer models. promote a pro-inflammatory tumor microenvironment. Cancer Res 2018;78: Genes Cancer 2014;5:261–72. 1752. 100. Clark AS, McAndrew NP, Troxel A, Feldman M, Lal P, Rosen M, et al. 114. Sorrentino JA, Lai AY, Strum JC, Roberts PJ. Trilaciclib (G1T28), a CDK4/6 Combination paclitaxel and palbociclib: results of a phase I trial in advanced inhibitor, enhances the efficacy of combination chemotherapy and immune breast cancer. Clin Cancer Res 2019;25:2072–9. checkpoint inhibitor treatment in preclinical models. Cancer Res 2017;77:5628.

1588 Mol Cancer Ther; 19(8) August 2020 MOLECULAR CANCER THERAPEUTICS

Chemotherapy and CDK4/6 Inhibitors: Unexpected Bedfellows

Patrick J. Roberts, Vishnu Kumarasamy, Agnieszka K. Witkiewicz, et al.

Mol Cancer Ther 2020;19:1575-1588. Published OnlineFirst June 16, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-18-1161

Cited articles This article cites 114 articles, 39 of which you can access for free at: http://mct.aacrjournals.org/content/19/8/1575.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/19/8/1575.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/19/8/1575. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.