Abstract PF-07220060 is a selective inhibitor of CDK4, displaying ~20-fold and ~4-fold increased selectivity for CDK4 versus CDK6 when compared to palbociclib and abemaciclib/ribociclib, respectively. This translates to less PF-07220060 associated neutropenia in beagle dogs and humans when compared to dual CDK4/6 inhibitors. Because neutropenia is the primary culprit necessitating limited and often interrupted clinical dosing of dual CDK4/6 inhibitors, the advantage of PF-07220060 lies in the flexibility to escalate the drug’s exposure in patients and thus realize near-complete target coverage of the CDK4 oncogene in tumors that are dependent on this kinase. Here we select tumor types/indications that show dependency on CDK4 but not CDK6 and includes cells derived from luminal breast cancer and androgen-receptor positive metastatic prostate cancer. Additionally, we investigate certain drugs with which PF-07220060 may be combined to maximize its efficacy in these tumor indications. PF-07220060 sensitizes HR+ HER2- breast cancer to the estrogen inhibitor, fulvestrant and the degrader, ARV-471 (vepdegestrant). PF-07220060 + vepdegestrant showed significantly longer tumor regrowth delay vs monotherapy groups and vs Palbo + vepdegestrant and vs PF-07220060 + fulvestrant*. Similarly, PF-07220060 sensitizes prostate cancer to the androgen receptor antagonist, enzalutamide. In HR+ HER2- breast cancer cells, the senescent cell fraction was markedly increased by co-treatment with PF-07220060 and fulvestrant versus either drug alone, albeit the combination did not trigger tumor cell death. Further addition of the PI3K inhibitor alpelisib was sufficient to enforce tumor shrinkage in vivo. Alternatively, inhibition of CDK4 plus CDK2 also led to tumor shrinkage in xenograft models of HR+, HER2- breast cancer. Finally, our preclinical data indicate that the propensity of CDK6 to compensate for CDK4 inhibition in these tumor types is limited. Compensation by CDK6 was seen in only a fraction of the evaluated in vitro and in vivo models. Even so, in these instances, PF-07220060’s efficacy remained comparable to palbociclib’s when both drugs were used at their therapeutic doses. We conclude that PF-07220060’s anti-tumor efficacy is broadly superior to currently approved dual CDK4/6 inhibitors. Citation Format: Lars Anders, Bernadette Pascual, Britton Boras, Julie Cianfrogna, Scott Garza, Na Li, Jing Tang Yuan, Mark Moen, Nanni Huser, Gary Gallego, Mehran Jalaie, Sacha Ninkovic, Sujin Cho-Schultz, Hong Shen, John Kath, Klaus Dress, Wade Diehl, Sajiv Nair, Rhys Jones, Jennifer Lafontaine, Anwar Murtaza, Aida Sacaan, Sudhakar Chintharlapalli, Todd VanArsdale. Preclinical development of the CDK4 selective inhibitor PF-07220060: Increased CDK4 versus CDK6 inhibition leads to improved anti-tumor efficacy at therapeutic concentrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 595.

Abstract The selective inhibition of Cyclin\CDK complexes controlling cell cycle progression has been established as cancer therapy by selective CDK4/6 inhibitors in HR+/HER2- breast cancer. Expanding control of the cell cycle through selective inhibition of CDK2 offers novel therapeutic opportunities in cancer, including targeting CCNE1 amplified tumors and countering resistance to CDK4\6 inhibitors in ER+ breast cancer. PF-07104091 is a first-in-class CDK2-selective inhibitor under clinical investigation in patients with HR+/HER2- breast cancer and ovarian cancer. Preclinical studies using PF-07104091 establish the therapeutic impact of CDK2 inhibition in both diseases and highlight distinct mechanistic roles for CDK2 in control of cancer cell proliferation. In models of CCNE1 amplified ovarian cancer, CDK2 plays the dominant role in control of RB1 phosphorylation and the G1 checkpoint. CDK2 inhibition with PF-07104091 induces G1 growth arrest and controls tumor xenograft growth as single agent therapy. In ER+ breast models CDK2 plays a supportive role in the control of RB1 phosphorylation cooperating with CDK4\6. Whole genome CRISPR KO and CRISPR activation screens in conjunction with CDK4\6 inhibition establish CDK2 KO as a primary sensitizer to CDK4\6 inhibition, and support Cyclin E\CDK2 complexes as the driver of resistance to CDK4\6 inhibitors in ER+ breast models. PF-07104091 combined with CDK4\6 inhibitor Palbociclib or CDK4-selective inhibitor PF-0060 synergistically controls proliferation of ER+ BC cells in vitro and induces tumor regression in ER+ BC xenograft models, including PDX models with acquired resistance to CDK4\6 inhibitors and endocrine therapy. Citation Format: Chen Shen, Ming Qiu, Nanni Huser, Bernadette Pascual, Qin Zhang, Todd VanArsdale, Anwar Murtaza, Jonathan Almaden, berly Kim, Lars Anders, Koleen Eisele. PF-07104091, a first-in-class CDK2-selective inhibitor for the treatment of HR+/HER2- breast cancer and CCNE1high ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5709.

The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.

A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. JDP2 encodes a bZIP protein that has been implicated as a T-ALL oncogene from insertional mutagenesis studies in mice, but its role in human T-ALL pathogenesis has remained obscure. Here we show that JDP2 is aberrantly expressed in a subset of T-ALL patients and is associated with poor survival. JDP2 is required for T-ALL cell survival, as its depletion by short hairpin RNA knockdown leads to apoptosis. Mechanistically, JDP2 regulates prosurvival signaling through direct transcriptional regulation of MCL1. Furthermore, JDP2 is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from rag2:jdp2 transgenic zebrafish express high levels of mcl1 and demonstrate resistance to steroids in vivo. These studies establish JDP2 as a novel oncogene in high-risk T-ALL and implicate overexpression of MCL1 as a mechanism of steroid resistance in JDP2-overexpressing cells.

Cyclins and cyclin-dependent kinases (CDKs) are hyperactivated in numerous human tumors. To identify means of interfering with cyclins/CDKs, we performed nine genome-wide screens for human microRNAs (miRNAs) directly regulating cell-cycle proteins. We uncovered a distinct class of miRNAs that target nearly all cyclins/CDKs, which are very effective in inhibiting cancer cell proliferation. By profiling the response of over 120 human cancer cell lines, we derived an expression-based algorithm that can predict the response of tumors to cell-cycle-targeting miRNAs. Using systemic administration of nanoparticle-formulated miRNAs, we inhibited tumor progression in seven mouse xenograft models, including three treatment-refractory patient-derived tumors, without affecting normal tissues. Our results highlight the utility of using cell-cycle-targeting miRNAs for treatment of refractory cancer types.

BET inhibitors that target bromodomain chromatin readers such as BRD4 are being explored as potential therapeutics in cancer; here triple-negative breast cancer cell lines are shown to respond to BET inhibitors and resistance seems to be associated with transcriptional changes rather than drug efflux and mutations, opening potential avenues to improve clinical responses to BET inhibitors. BET inhibitors that target bromodomain chromatin readers such as BRD4 are being explored as potential therapeutics in cancer. Here Kornelia Polyak and colleagues investigate the response of breast cancer cell lines and xenograft mouse models to BET inhibitors. They find that triple-negative breast cancer cell lines respond to BET inhibitors. Resistance can emerge, but there is no evidence for mechanisms involving drug efflux or mutations in the bromodomain genes or known driver genes. Instead, there are transcriptional changes and increased recruitment of BRD4 to chromatin independent of its bromodomain, concomitant with its increased phosphorylation. Together with two recent Nature publications from the laboratories of Mark Dawson and Johannes Zuber dealing with different cancers, the study suggests potential avenues to improve clinical responses to BET inhibitors. Jeff Settleman discusses all three papers in News & Views. Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy1,2,3. BET bromodomain inhibitors, which have shown efficacy in several models of cancer4,5,6, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs7,8,9. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.

Neuroblastoma is an embryonal tumor of the peripheral sympathetic nervous system, accounting for 15% of all childhood cancer deaths. Both overexpression of the transcription factor LMO1 and the polymorphisms within this gene locus are associated with the susceptibility to neuroblastoma, but the oncogenic roles of LMO1 in neuroblastoma pathogenesis have not been elucidated. The roles of LMO1 in T-cell acute lymphoblastic leukemia (T-ALL) are better understood, and these suggest that some of its effects may be similar between the two malignancies. Here we identify the transcriptional regulatory program controlled by LMO1 in neuroblastoma and T-ALL cells. Knockdown of LMO1 induces apoptotic cell death in both tumor types. ChIP-seq and microarray analyses demonstrate that LMO1 frequently co-occupies its target genes with GATA3 and regulates gene expression in a tissue-specific manner. LMO1 positively regulates genes involved in neuronal development, tumor invasion and metastasis in neuroblastoma cells, whereas it regulates genes involved in lymphopoiesis and immune function in T-ALL cells. Gene set enrichment analysis reveals that many genes bound by LMO1 and GATA3 are significantly downregulated upon MYCN knockdown in the MYCN-amplified neuroblastoma cells. Importantly, LMO1 binds at the CDK6 gene locus, which is associated with a super-enhancer both in neuroblastoma and T-ALL cells. The mRNA expression of CDK6 is positively correlated with LMO1 expression in primary neuroblastoma samples. Knockdown of LMO1 downregulates CDK6 protein expression, whereas overexpression of LMO1 upregulates CDK6 expression. CDK6 knockdown induces apoptosis both in neuroblastoma and T-ALL cells. Our results indicate that CDK6 is a critical downstream target that is directly activated by LMO1 and is required for cell survival in neuroblastoma and T-ALL cells. Citation Format: Takaomi Sanda, Koshi Akahanse, Brian J. Abraham, Nina Weichert, Adam Durbin, Lars Anders, Shi Hao Tan, Alice Wei Yee Yam, Lee N. Lawton, Richard A. Young, John M. Maris, A Thomas Look. Transcriptional regulatory program controlled by the oncogenic transcription factor LMO1 in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2007.

Many transcription factors, chromatin-associated proteins and regulatory DNA elements are genetically and/or epigenetically altered in cancer, including Chronic Myeloid Leukemia (CML). This leads to deregulation of transcription that is often causally linked to the tumorigenic state. Chromatin-immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is the key technology to study transcription as it allows in vivo whole-genome mapping of epigenetic modifications and interactions of proteins with DNA or chromatin. However, numerous DNA/chromatin-binding proteins, including EZH2, remain difficult to "ChIP," thus yielding genome-wide binding maps of only suboptimal quality. Here, we describe a ChIP-seq protocol optimized for high-quality protein-genome binding maps that have proven especially useful for studying difficult to 'ChIP' transcription regulatory factors in Chronic Myeloid Leukemia (CML) and related malignancies.

causal variant is identified at the LMO1 oncogene locus that drives the genetic association of LMO1 with neuroblastoma susceptibility; the causal SNP disrupts a GATA transcription factor binding site within a tissue-specific super-enhancer element in the first intron of LMO1 , thereby affecting LMO1 expression.

In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, that recruit much of the cell’s transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits its H3K27 acetylase–binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, which suggests a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells.

Abstract A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide new and important insights into the mechanisms by which such small molecules interfere with tumor cell functions. We have developed a method that couples affinity capture of chemical entities and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small molecules throughout the human genome. Using Chem-seq, we have uncovered the full repertoire of the genomic sites bound by a BET bromodomain inhibitor, a cyclin-dependent kinase (CDK) inhibitor and a DNA intercalating drug. Moreover, by combining Chem-seq with ChIP-seq, we have characterized the interactions of drugs with their targets throughout the genome of tumor cells. These methods provide a powerful approach to enhance understanding of therapeutic action and characterize the specificity of drugs that interact with DNA or genome-associated proteins. Citation Format: Lars Anders, Matthew G. Guenther, Jun Qi, Zi Peng Fan, Jason J. Marineau, Peter B. Rahl, Jakob Lovén, Alla A. Sigova, William B. Smith, Tong Ihn Lee, James E. Bradner, Richard A. Young. Genome-wide localization of anti-cancer drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3230. doi:10.1158/1538-7445.AM2014-3230

D-type cyclins (D1, D2, and D3) are components of the mammalian core cell-cycle machinery and function to drive cell proliferation. Here, we report that D-cyclins perform a rate-limiting antiapoptotic function in vivo. We found that acute shutdown of all three D-cyclins in bone marrow of adult mice resulted in massive apoptosis of all hematopoietic cell types. We demonstrate that adult hematopoietic stem cells are particularly dependent on D-cyclins for survival and that they are especially sensitive to cyclin D loss. Surprisingly, we found that the antiapoptotic function of D-cyclins also operates in quiescent hematopoietic stem and progenitor cells. Our analyses revealed that D-cyclins repress the expression of the death receptor Fas and its ligand, FasL. Acute ablation of D-cyclins upregulated these proapoptotic genes and led to Fas- and caspase 8-dependent apoptosis. These results reveal an unexpected function of cell-cycle proteins in controlling apoptosis in normal cell homeostasis.

Hepatic gluconeogenesis is crucial to maintain normal blood glucose during periods of nutrient deprivation. Gluconeogenesis is controlled at multiple levels by a variety of signal transduction and transcriptional pathways. However, dysregulation of these pathways leads to hyperglycemia and type 2 diabetes. While the effects of various signaling pathways on gluconeogenesis are well established, the downstream signaling events repressing gluconeogenic gene expression are not as well understood. The cell-cycle regulator cyclin D1 is expressed in the liver, despite the liver being a quiescent tissue. The most well-studied function of cyclin D1 is activation of cyclin-dependent kinase 4 (CDK4), promoting progression of the cell cycle. We show here a novel role for cyclin D1 as a regulator of gluconeogenic and oxidative phosphorylation (OxPhos) gene expression. In mice, fasting decreases liver cyclin D1 expression, while refeeding induces cyclin D1 expression. Inhibition of CDK4 enhances the gluconeogenic gene expression, whereas cyclin D1–mediated activation of CDK4 represses the gluconeogenic gene-expression program in vitro and in vivo. Importantly, we show that cyclin D1 represses gluconeogenesis and OxPhos in part via inhibition of peroxisome proliferator–activated receptor γ coactivator-1α (PGC1α) activity in a CDK4-dependent manner. Indeed, we demonstrate that PGC1α is novel cyclin D1/CDK4 substrate. These studies reveal a novel role for cyclin D1 on metabolism via PGC1α and reveal a potential link between cell-cycle regulation and metabolic control of glucose homeostasis.

National Institutes of Health (U.S.) (Grants CA98543, CA114766, CA98413, CA30969 and CA29139)

Research over the past quarter century has identified cyclin D-dependent kinases, CDK4 and CDK6, as the major oncogenic drivers among members of the CDK superfamily. CDK4/6 are rendered hyperactive in the majority of human cancers through a multitude of genomic alterations. Sustained activation of these protein kinases provides cancer cells with the power to enter the cell cycle continuously by triggering G1-S-phase transitions and dramatically shortening the duration of the G1 phase. It has also become clear, however, that CDK4/6 effectively counter cancer cell-intrinsic tumor suppression mechanisms, senescence and apoptosis, which must be overcome during cell transformation and kept at bay throughout all stages of tumorigenesis. As a central ‘node’ in cellular signaling networks, cyclin D-dependent kinases sense a plethora of mitogenic signals to orchestrate specific transcriptional programs. As the complexity of the cellular signaling network regulated by these oncogenic kinases unfolds, much remains to be learned about its architecture, its dynamics and the consequences of its perturbation.

A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chemical entities that interact with DNA or genome-associated proteins.

Cyclin D-dependent kinases (CDK4 and CDK6) are positive regulators of cell cycle entry and they are overactive in the majority of human cancers. However, it is currently not completely understood by which cellular mechanisms CDK4/6 promote tumorigenesis, largely due to the limited number of identified substrates. Here we performed a systematic screen for substrates of cyclin D1-CDK4 and cyclin D3-CDK6. We identified the Forkhead Box M1 (FOXM1) transcription factor as a common critical phosphorylation target. CDK4/6 stabilize and activate FOXM1, thereby maintain expression of G1/S phase genes, suppress the levels of reactive oxygen species (ROS), and protect cancer cells from senescence. Melanoma cells, unlike melanocytes, are highly reliant on CDK4/6-mediated senescence suppression, which makes them particularly susceptible to CDK4/6 inhibition.

Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTPkappa is cleaved by three proteases: furin, ADAM 10, and gamma-secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTPkappa. This is followed by gamma-secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates beta-catenin, an RPTPkappa substrate. The expression of RPTPkappa suppresses beta-catenin's transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of beta-catenin signaling. We also found that RPTPmu, another MAM family member, undergoes gamma-secretase-dependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTPkappa signaling and implicate PIC in the activation of beta-catenin-mediated transcription.