The tumor microenvironment (TME) profoundly influences tumorigenesis, with gene expression in the breast TME capable of predicting clinical outcomes. The TME is complex and includes distinct cancer-associated fibroblast (CAF) subtypes whose contribution to tumorigenesis remains unclear. Here, we identify a subset of myofibroblast CAFs (myCAF) that are senescent (senCAF) in mouse and human breast tumors. Utilizing the MMTV-PyMT;INK-ATTAC (INK) mouse model, we found that senCAF-secreted extracellular matrix specifically limits natural killer (NK) cell cytotoxicity to promote tumor growth. Genetic or pharmacologic senCAF elimination unleashes NK cell killing, restricting tumor growth. Finally, we show that senCAFs are present in HER2+, ER+, and triple-negative breast cancer and in ductal carcinoma in situ (DCIS) where they predict tumor recurrence. Together, these findings demonstrate that senCAFs are potently tumor promoting and raise the possibility that targeting them by senolytic therapy could restrain breast cancer development. Significance: senCAFs limit NK cell-mediated killing, thereby contributing to breast cancer progression. Thus, targeting senCAFs could be a clinically viable approach to limit tumor progression. See related article by Belle et al., p. 1324.

Progression through G1/S phase of the cell cycle is coordinated by cyclin-dependent kinase (CDK) activities. Here, we find that the requirement for different CDK activities and cyclins in driving cancer cell cycles is highly heterogeneous. The differential gene requirements associate with tumor origin and genetic alterations. We define multiple mechanisms for G1/S progression in RB-proficient models, which are CDK4/6 independent and elicit resistance to FDA-approved inhibitors. Conversely, RB-deficient models are intrinsically CDK4/6 independent, but exhibit differential requirements for cyclin E. These dependencies for CDK and cyclins associate with gene expression programs that denote intrinsically different cell-cycle states. Mining therapeutic sensitivities shows that there are reciprocal vulnerabilities associated with RB1 or CCND1 expression versus CCNE1 or CDKN2A. Together, these findings illustrate the complex nature of cancer cell cycles and the relevance for precision therapeutic intervention.

The retinoblastoma tumor suppressor gene (RB1) plays a critical role in coordinating multiple pathways that impact cancer initiation, disease progression, and therapeutic responses. Here we probed molecular features associated with the RB-pathway across 31 tumor-types. While the RB-pathway has been purported to exhibit multiple mutually exclusive genetic events, only RB1 alteration is mutually exclusive with deregulation of CDK4/6 activity. An ER+ breast cancer model with targeted RB1 deletion was used to identify signatures of CDK4/6 activity and RB-dependency (CDK4/6-RB integrated signature). This signature was prognostic in tumor-types with gene expression features indicative of slower growth. Single copy loss on chromosome 13q encompassing the RB1 locus is prevalent in many cancers, yielding reduced expression of multiple genes in cis, and is inversely related to the CDK4/6-RB integrated signature supporting a cause-effect relationship. Genes that are positively and inversely correlated with the CDK4/6-RB integrated signature define new tumor-specific pathways associated with RB-pathway activity.

Intrinsic or acquired resistance to clinically approved CDK4/6 inhibitors has emerged as a major obstacle that hinders their utility beyond ER+ breast cancer. In this study, CDK4/6-dependent and -resistant models were employed to identify functional determinants of response to pharmacologic CDK4/6 inhibitors. In all models tested, the activation of RB and inhibition of CDK2 activity emerged as determinants of sensitivity. While depleting CDK4 and 6 was sufficient to limit proliferation in specific resistance settings, RB loss rendered cells completely independent of these kinases. The main downstream target in this context was the activation status of CDK2, which was suppressed with CDK4/6 inhibition in an RB-dependent fashion. Protein levels of p27 were associated with plasticity/rigidity of the cell cycle and correlated with sensitivity to CDK4/6 inhibition. Exogenous overexpression and pharmacologic induction of p27 via inhibition of SKP2 and targeting the MEK/ERK pathway enhanced the cytostatic effect of CDK4/6 inhibitors. Mice bearing ER+ xenografts displayed a durable antitumor response to palbociclib; however, over the course of treatment, few cells retained RB phosphorylation, which was associated with limited p27 protein levels as determined by multispectral imaging. Similarly, combination treatment of palbociclib with a MEK inhibitor in pancreatic cancer PDX models upregulated p27 and further enhanced the in vivo tumor response to palbociclib. Collectively, these results suggest that the cell cycle plasticity, which enables tumor models to evade palbociclib-mediated activation of RB, could be targeted using a clinically applicable CDK2 inhibitor. Significance: This work provides a mechanistic insight toward understanding the functional roles of multiple cell cycle regulators that drive plasticity and sensitivity to CDK4/6 inhibition.

Objective This study exploits the intersection between molecular-targeted therapies and immune-checkpoint inhibition to define new means to treat pancreatic cancer. Design Patient-derived cell lines and xenograft models were used to define the response to CDK4/6 and MEK inhibition in the tumour compartment. Impacts relative to immunotherapy were performed using subcutaneous and orthotopic syngeneic models. Single-cell RNA sequencing and multispectral imaging were employed to delineate effects on the immunological milieu in the tumour microenvironment. Results We found that combination treatment with MEK and CDK4/6 inhibitors was effective across a broad range of PDX models in delaying tumour progression. These effects were associated with stable cell-cycle arrest, as well as the induction of multiple genes associated with interferon response and antigen presentation in an RB-dependent fashion. Using single-cell sequencing and complementary approaches, we found that the combination of CDK4/6 and MEK inhibition had a significant impact on increasing T-cell infiltration and altering myeloid populations, while potently cooperating with immune checkpoint inhibitors. Conclusions Together, these data indicate that there are canonical and non-canonical features of CDK4/6 and MEK inhibition that impact on the tumour and immune microenvironment. This combination-targeted treatment can promote robust tumour control in combination with immune checkpoint inhibitor therapy.