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The combination of CDK4/6 and MEK inhibition as a therapeutic strategy has shown promise in various cancer models, particularly in those harboring RAS mutations. An initial high-throughput drug screen identified a high synergy between the CDK4/6 inhibitor palbociclib and the MEK inhibitor trametinib when used in combination in soft tissue sarcomas. In RAS mutant models, combination treatment with palbociclib and trametinib induced significant G1 cell cycle arrest, resulting in a marked reduction in cell proliferation and growth. CRISPR-mediated RB1 depletion resulted in a decreased response to CDK4/6 and MEK inhibition, which was validated in both cell culture and xenograft models. Beyond its cell cycle inhibitory effects, pathway enrichment analysis revealed the robust activation of interferon pathways upon CDK4/6 and MEK inhibition. This induction of gene expression was associated with the upregulation of retroviral elements. The TBK1(TANK-binding kinase 1) inhibitor GSK8612 selectively blocked the induction of interferon-related genes induced by palbociclib and trametinib treatment, and highlighted the separable epigenetic responses elicited by combined CDK4/6 and MEK inhibition. Together, these findings provide key mechanistic insights into the therapeutic potential of CDK4/6 and MEK inhibition in soft tissue sarcoma.

In the commonly accepted paradigm for control of the mammalian cell cycle, sequential cyclin-dependent kinase (CDK) and cyclin activities drive the orderly transition from G1 to S phase. However, recent studies using different technological approaches and examining a broad range of cancer cell types are challenging this established paradigm. An alternative model is evolving in which cell cycles utilize different drivers and take different trajectories through the G1/S transition. We are discovering that cancer cells in particular can adapt their drivers and trajectories, which has important implications for antiproliferative therapies. These studies have helped to refine an understanding of how CDK inhibition impinges on proliferation and have significance for understanding fundamental features of cell biology and cancer., Competing Interests: Declaration of interests E.S.K receives research funding from Bristol Meyer Squibb and Blueprint Medicine. He is a member of the consulting organization Cancer Cell Cycles-LLC. A.K.W. receives research funding from Bristol Meyer Squibb and Blueprint Medicine. S.M.R. receives research funding from Type6 Therapeutics., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (©2024 American Association for Cancer Research.)

The largest portion of breast cancer patients diagnosed after 70 years of age present with hormone receptor-positive (HR+) breast cancer subtypes. Cyclin-dependent kinase (CDK) 4/6 inhibitor treatment, in conjunction with endocrine therapy, has become standard-of-care for metastatic HR+ breast cancer. In total, 320 patients with metastatic breast cancer receiving CDK4/6 inhibitor combined with fulvestrant or an aromatase inhibitor were enrolled in an ongoing observational study or were included in an IRB-approved retrospective study. All patients receiving CDK4/6 inhibitor-based therapy that were ≥70 years of age (n = 111) displayed prolonged progression-free survival (27.6 months) as compared to patients <70 years of age (n = 209, 21.1 months, HR = 1.38, p < 0.05). Specifically, patients receiving a CDK4/6 inhibitor with an aromatase inhibitor who were ≥70 years of age (n = 79) displayed exceptionally prolonged progression-free survival (46.0 months) as compared to patients receiving the same treatment who were <70 years of age (n = 161, 21.8 months, HR = 1.71, p < 0.01). However, patients ≥70 years of age also experienced more frequent adverse responses to CDK4/6 inhibitor-based treatment leading to dose reduction, hold, or discontinuation than the younger cohort (69% and 53%, respectively). Treatment strategies that may decrease toxicity without affecting efficacy (such as dose titration) are worth further exploration.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that lacks effective treatment options, highlighting the need for developing new therapeutic interventions. Here, we assessed the response to pharmacologic inhibition of KRAS, the central oncogenic driver of PDAC. In a panel of PDAC cell lines, inhibition of KRASG12D with MRTX1133 yielded variable efficacy in suppressing cell growth and downstream gene expression programs in 2D cultures. On the basis of CRISPR-Cas9 loss-of-function screens, ITGB1 was identified as a target to enhance the therapeutic response to MRTX1133 by regulating mechanotransduction signaling and YAP/TAZ expression, which was confirmed by gene-specific knockdown and combinatorial drug synergy. Interestingly, MRTX1133 was considerably more efficacious in 3D cell cultures. Moreover, MRTX1133 elicited a pronounced cytostatic effect in vivo and controlled tumor growth in PDAC patient-derived xenografts. In syngeneic models, KRASG12D inhibition led to tumor regression that did not occur in immune-deficient hosts. Digital spatial profiling on tumor tissues indicated that MRTX1133-mediated KRAS inhibition enhanced IFNγ signaling and induced antigen presentation that modulated the tumor microenvironment. Further investigation of the immunologic response using single-cell sequencing and multispectral imaging revealed that tumor regression was associated with suppression of neutrophils and influx of effector CD8+ T cells. Together, these findings demonstrate that both tumor cell-intrinsic and -extrinsic events contribute to response to MRTX1133 and credential KRASG12D inhibition as a promising therapeutic strategy for a large percentage of patients with PDAC., Significance: Pharmacologic inhibition of KRAS elicits varied responses in pancreatic cancer 2D cell lines, 3D organoid cultures, and xenografts, underscoring the importance of mechanotransduction and the tumor microenvironment in regulating therapeutic responses., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibition yields differential cellular responses in multiple tumor models due to redundancy in cell cycle. We investigate whether the differential requirements of CDKs in multiple cell lines function as determinant of response to pharmacological agents that target these kinases., Methods: We utilized proteolysis-targeted chimeras (PROTACs) that are conjugated with palbociclib (Palbo-PROTAC) to degrade both CDK4 and CDK6. FN-POM was synthesized by chemically conjugating pomalidomide moiety with a multi-kinase inhibitor, FN-1501. Patient derived PDAC organoids and PDX model were utilized to investigate the effect of FN-POM in combination with palbociclib., Results: Palbo-PROTAC mediates differential impact on cell cycle in different tumor models, indicating that the dependencies to CDK4 and 6 kinases are heterogenous. Cyclin E overexpression uncouples cell cycle from CDK4/6 and drives resistance to palbo-PROTAC. Elevated expression of P16INK4A antagonizes PROTAC-mediated degradation of CDK4 and 6. FN-POM degrades cyclin E and CDK2 and inhibits cell cycle progression in P16INK4A-high tumor models. Combination of palbociclib and FN-POM cooperatively inhibit tumor cell proliferation via RB activation., Conclusion: Resistance to CDK4/6 inhibition could be overcome by pharmacologically limiting Cyclin E/CDK2 complex and proves to be a potential therapeutic approach., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Despite widespread use and a known mechanism of action for CDK4/6 inhibitors in combination with endocrine therapy, features of disease evolution and determinants of therapeutic response in the real-world setting remain unclear. Here, a cohort of patients treated with standard-of-care combination regimens was utilized to explore features of disease and determinants of progression-free survival (PFS) and overall survival (OS). In this cohort of 280 patients, >90% of patients were treated with palbociclib in combination with either an aromatase inhibitor (AI) or fulvestrant (FUL). Most of these patients had modified Scarff-Bloom-Richardson (SBR) scores, and ER, HER2, and PR immunohistochemistry. Both the SBR score and lack of PR expression were associated with shorter PFS in patients treated with AI combinations and remained significant in multivariate analyses (HR = 3.86, p = 0.008). Gene expression analyses indicated substantial changes in cell cycle and estrogen receptor signaling during the course of treatment. Furthermore, gene expression-based subtyping indicated that predominant subtypes changed with treatment and progression. The luminal B, HER2, and basal subtypes exhibited shorter PFS in CDK4/6 inhibitor combinations when assessed in the pretreatment biopsies; however, they were not associated with OS. Using unbiased approaches, cell cycle-associated gene sets were strongly associated with shorter PFS in pretreatment biopsies irrespective of endocrine therapy. Estrogen receptor signaling gene sets were associated with longer PFS particularly in the AI-treated cohort. Together, these data suggest that there are distinct pathological and biological features of HR+/HER2- breast cancer associated with response to CDK4/6 inhibitors. Clinical trial registration number: NCT04526587., (© 2023. Nature Publishing Group UK.)

Anti-estrogens or aromatase inhibitors in combination with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are the current standard of care for estrogen receptor-positive (ER+) Her-2 negative metastatic breast cancer. Although these combination therapies prolong progression-free survival compared to endocrine therapy alone, the growth-arrested state of residual tumor cells is clearly transient. Tumor cells that escape what might be considered a dormant or quiescent state and regain proliferative capacity often acquire resistance to further therapies. Our studies are based upon the observation that breast tumor cells arrested by Fulvestrant + Palbociclib enter into states of both autophagy and senescence from which a subpopulation ultimately escapes, potentially contributing to recurrent disease. Autophagy inhibition utilizing pharmacologic or genetic approaches only moderately enhanced the response to Fulvestrant + Palbociclib in ER+ MCF-7 breast tumor cells, slightly delaying proliferative recovery. In contrast, the BET inhibitor/degrader, ARV-825, prolonged the growth arrested state in both p53 wild type MCF-7 cells and p53 mutant T-47D cells and significantly delayed proliferative recovery. In addition, ARV-825 added after the Fulvestrant + Palbociclib combination promoted apoptosis and demonstrated efficacy in resistant RB deficient cell lines. These studies indicate that administration of BET inhibitors/degraders, which are currently being investigated in multiple clinical trials, may potentially improve standard of care therapy in metastatic ER+ breast cancer patients and may further prolong progression-free survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Finnegan, Elshazly, Patel, Tyutyunyk-Massey, Tran, Kumarasamy, Knudsen and Gewirtz.)

Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent "long-tail" breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 ("EpiDrivers"), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in ∼39% of human breast cancers, and ∼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology., Significance: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711., (©2022 American Association for Cancer Research.)

The mammalian cell cycle has been extensively studied regarding cancer etiology, progression, and therapeutic intervention. The canonical cell cycle framework is supported by a plethora of data pointing to a relatively simple linear pathway in which mitogenic signals are integrated in a stepwise fashion to allow progression through G1/S with coordinate actions of cyclin-dependent kinases (CDK)4/6 and CDK2 on the RB tumor suppressor. Recent work on adaptive mechanisms and intrinsic heterogeneous dependencies indicates that G1/S control of the cell cycle is a variable signaling pathway rather than an invariant engine that drives cell division. These alterations can limit the effectiveness of pharmaceutical agents but provide new avenues for therapeutic interventions. These findings support a dystopian view of the cell cycle in cancer where the canonical utopian cell cycle is often not observed. However, recognizing the extent of cell cycle heterogeneity likely creates new opportunities for precision therapeutic approaches specifically targeting these states., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: A study was initiated at Roswell Park Comprehensive Cancer Center to capture the real-world experience related to the use of CDK4/6 inhibitors (Ciclibs) for the treatment of metastatic hormone receptor-positive and HER2-negative breast cancer (HR+/HER2-)., Patients and Methods: A total of 222 patients were evaluated who received CDK4/6 inhibitors in the period from 2015 to 2021. Detailed clinical and demographic information was obtained on each patient and used to define clinical and demographic features associated with progression-free survival on CDK4/6 inhibitor-based therapies., Results: In this real-world analysis, the majority of patients received palbociclib as the CDK4/6 inhibitor with letrozole or fulvestrant as the predominant endocrine therapies. The median progression-free survival (PFS) in the letrozole (27.6 months) and fulvestrant (17.2 months) groups were comparable to that observed in clinical trials. As expected, age at start of the treatment and menopausal status influenced endocrine therapy utilization but were not associated with PFS. Patients with recurrent disease had shorter PFS (P = .0024) than those presenting with de novo metastasis. The presence of visceral metastasis trended toward shorter PFS (P = .051). Similarly, prior endocrine therapy (P = .003) or chemotherapy (P = .036) was associated with shorter PFS. Body mass index was not associated with PFS or with dose interruption and/or modification. While the number of minorities in this analysis is limited (n = 26), these patients as a group had statistically shorter PFS on treatment (P = .002)., Conclusions: The real-world progression-free survival with CDK4/6 inhibitors mimics that observed in the clinical trial. A number of clinical and demographic features were associated with PFS on CDK4/6 inhibitor-based therapy. Further studies are ongoing to validate these findings incorporating additional cancer centers., (© The Author(s) 2022. Published by Oxford University Press.)

The management of metastatic estrogen receptor (ER) positive HER2 negative breast cancer (ER+) has improved; however, therapeutic resistance and disease progression emerges in majority of cases. Using unbiased approaches, as expected PI3K and MTOR inhibitors emerge as potent inhibitors to delay proliferation of ER+ models harboring PIK3CA mutations. However, the cytostatic efficacy of these drugs is hindered due to marginal impact on the expression of cyclin D1. Different combination approaches involving the inhibition of ER pathway or cell cycle result in durable growth arrest via RB activation and subsequent inhibition of CDK2 activity. However, cell cycle alterations due to RB loss or ectopic CDK4/cyclin D1 activation yields resistance to these cytostatic combination treatments. To define means to counter resistance to targeted therapies imparted with RB loss; complementary drug screens were performed with RB-deleted isogenic cell lines. In this setting, RB loss renders ER+ breast cancer models more vulnerable to drugs that target DNA replication and mitosis. Pairwise combinations using these classes of drugs defines greater selectivity for RB deficiency. The combination of AURK and WEE1 inhibitors, yields synergistic cell death selectively in RB-deleted ER+ breast cancer cells via apoptosis and yields profound disease control in vivo. Through unbiased efforts the XIAP/CIAP inhibitor birinapant was identified as a novel RB-selective agent. Birinapant further enhances the cytotoxic effect of chemotherapies and targeted therapies used in the treatment of ER+ breast cancer models selectively in the RB-deficient setting. Using organoid culture and xenograft models, we demonstrate the highly selective use of birinapant based combinations for the treatment of RB-deficient tumors. Together, these data illustrate the critical role of RB-pathway in response to many agents used to treat ER+ breast cancer, whilst informing new therapeutic approaches that could be deployed against resistant disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Research documents social and economic antecedents of adverse birth outcomes, which may include involuntary job loss. Previous work on job loss and adverse birth outcomes, however, lacks high-quality individual data on, and variation in, plausibly exogenous job loss during pregnancy and therefore cannot rule out strong confounding., Methods: We analysed unique linked registries in Denmark, from 1980 to 2017, to examine whether a father's involuntary job loss during his spouse's pregnancy increases the risk of a low-weight (i.e. <2500 grams) and/or preterm (i.e. <37 weeks of gestational age) birth. We applied a matched-sibling design to 743 574 sibling pairs., Results: Results indicate an increased risk of a low-weight birth among infants exposed in utero to fathers' unexpected job loss [odds ratio (OR) = 1.37, 95% confidence interval (CI): 1.07, 1.75]. Sex-specific analyses show that this result holds for males (OR = 1.70, 95% CI: 1.14, 2.53) but not females (OR = 1.24, 95% CI: 0.80, 1.91). We find no relation with preterm birth., Conclusions: Findings support the inference that a father's unexpected job loss adversely affects the course of pregnancy, especially among males exposed in utero., (© The Author(s) 2021; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Studies have shown that Nrf2 E79Q/+ is one of the most common mutations found in human tumors. To elucidate how this genetic change contributes to lung cancer, we compared lung tumor development in a genetically-engineered mouse model (GEMM) with dual Trp53/p16 loss, the most common mutations found in human lung tumors, in the presence or absence of Nrf2 E79Q/+ . Trp53/p16-deficient mice developed combined-small cell lung cancer (C-SCLC), a mixture of pure-SCLC (P-SCLC) and large cell neuroendocrine carcinoma. Mice possessing the LSL-Nrf2 E79Q mutation showed no difference in the incidence or latency of C-SCLC compared with Nrf2 +/+ mice. However, these tumors did not express NRF2 despite Cre-induced recombination of the LSL-Nrf2 E79Q allele. Trp53/p16-deficient mice also developed P-SCLC, where activation of the NRF2 E79Q mutation associated with a higher incidence of this tumor type. All C-SCLCs and P-SCLCs were positive for NE-markers, NKX1-2 (a lung cancer marker) and negative for P63 (a squamous cell marker), while only P-SCLC expressed NRF2 by immunohistochemistry. Analysis of a consensus NRF2 pathway signature in human NE + -lung tumors showed variable activation of NRF2 signaling. Our study characterizes the first GEMM that develops C-SCLC, a poorly-studied human cancer and implicates a role for NRF2 activation in SCLC development., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: Declaration of interests A.L.W. has received royalties from licenses of patient-derived xenograft or organoid models issued by the University of Utah. The university may issue new licenses in the future at its discretion, which may result in additional royalties. E.S.K. and A.K.W. have served on the BioVica scientific advisory board., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: We evaluated the antitumor efficacy of cetuximab in combination with pembrolizumab in patients with RAS wild-type (RASwt), metastatic colorectal adenocarcinoma (mCRC)., Patients and Methods: In this phase Ib/II study, cetuximab was combined with pembrolizumab in patients with RASwt mCRC with ≥ one prior line of therapy for advanced disease. We analyzed baseline on-treatment tumor tissues for changes in the tumor microenvironment (TME), using flow cytometry and multispectral immunofluorescence., Results: Forty-four patients were evaluable for efficacy. The study was negative for the primary efficacy endpoint [overall response rate: 2.6%, 6-month progression-free survival (PFS): 31%; P = 0.52]. Median PFS was 4.1 months [95% confidence interval (CI): 3.9-5.5 months]. No increase in adverse effects was identified. We observed favorable immunomodulation with 47% increase in the number of intratumoral CTLs posttreatment ( P = 0.035). These changes were more pronounced in patients with tumor shrinkage ( P = 0.05). The TME was characterized by high numbers of TIM3 + and CTLA4 + cells; there were few activated OX40 + cells. PD-L1 expression was higher in pretreatment tumor cells from metastatic sites versus primary tumor samples ( P < 0.05). Higher numbers of PD-L1 + tumor cells at baseline were associated with tumor shrinkage ( P = 0.04). Analysis of immune populations in the blood demonstrated decreases in PD-1 + memory effector cells ( P = 0.04) and granulocytic myeloid-derived suppressor cells ( P = 0.03), with simultaneous increases in CD4 + /CTLA4 + cells ( P = 0.01)., Conclusions: The combination of cetuximab and pembrolizumab is inactive in patients with RASwt mCRC, despite its partial local immunologic efficacy. Further development of immuno-oncology combinations with enhanced efficacy and/or targeting additional or alternative immune checkpoints merits investigation., (©2021 American Association for Cancer Research.)

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAP on or YAP off cancer classes that express or silence YAP, respectively. YAP off solid cancers are neural/neuroendocrine and frequently RB1 -/- , such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAP off or YAP on cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications., Competing Interests: Declaration of interests B.H.L. reports honoraria and non-financial support from AstraZeneca, and has received research funding from Pfizer and AstraZeneca not related to this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (©2020 American Association for Cancer Research.)

Purpose: Increased β-adrenergic receptor (β-AR) signaling has been shown to promote the creation of an immunosuppressive tumor microenvironment (TME). Preclinical studies have shown that abrogation of this signaling pathway, particularly β2-AR, provides a more favorable TME that enhances the activity of anti-PD-1 checkpoint inhibitors. We hypothesize that blocking stress-related immunosuppressive pathways would improve tumor response to immune checkpoint inhibitors in patients. Here, we report the results of dose escalation of a nonselective β-blocker (propranolol) with pembrolizumab in patients with metastatic melanoma., Patients and Methods: A 3 + 3 dose escalation study for propranolol twice a day with pembrolizumab (200 mg every 3 weeks) was completed. The primary objective was to determine the recommended phase II dose (RP2D). Additional objectives included safety, antitumor activity, and biomarker analyses. Responders were defined as patients with complete or partial response per immune-modified RECIST at 6 months., Results: Nine patients with metastatic melanoma received increasing doses of propranolol in cohorts of 10, 20, and 30 mg twice a day. No dose-limiting toxicities were observed. Most common treatment-related adverse events (TRAEs) were rash, fatigue, and vitiligo, observed in 44% patients. One patient developed two grade ≥3 TRAEs. Objective response rate was 78%. While no significant changes in treatment-associated biomarkers were observed, an increase in IFNγ and a decrease in IL6 was noted in responders., Conclusions: Combination of propranolol with pembrolizumab in treatment-naïve metastatic melanoma is safe and shows very promising activity. Propranolol 30 mg twice a day was selected as RP2D in addition to pembrolizumab based on safety, tolerability, and preliminary antitumor activity., (©2020 American Association for Cancer Research.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Cyclin-dependent kinases 4 and 6 (CDK4/6) have emerged as important therapeutic targets. Pharmacologic inhibitors of these kinases function to inhibit cell-cycle progression and exert other important effects on the tumor and host environment. Because of their impact on the cell cycle, CDK4/6 inhibitors (CDK4/6i) have been hypothesized to antagonize the antitumor effects of cytotoxic chemotherapy in tumors that are CDK4/6 dependent. However, there are multiple preclinical studies that illustrate potent cooperation between CDK4/6i and chemotherapy. Furthermore, the combination of CDK4/6i and chemotherapy is being tested in clinical trials to both enhance antitumor efficacy and limit toxicity. Exploitation of the noncanonical effects of CDK4/6i could also provide an impetus for future studies in combination with chemotherapy. Thus, while seemingly mutually exclusive mechanisms are at play, the combination of CDK4/6 inhibition and chemotherapy could exemplify rational medicine., (©2020 American Association for Cancer Research.)

Purpose: Metastatic pancreatic adenocarcinoma (mPC) has a poor prognosis. CDK4/6 is often deregulated in mPC due to CDKN2A loss, resulting in the loss of p16INK4a that inhibits CDK4/6. CDK4/6 inhibitor monotherapy is ineffective due to RAS-mediated activation of alternative pathways, including phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR). We conducted a phase I study combining CDK4/6 and mTOR inhibition in patients with mPC refractory to standard chemotherapy. Materials and Methods: The combination of ribociclib (a CDK4/6 inhibitor) and everolimus (an mTOR inhibitor) was investigated in a phase I study in patients with mPC and progression on 5-fluorouracil- and gemcitabine-based chemotherapy. A 3 + 3 design was used to find the recommended phase II dose (RP2D) of ribociclib (250 or 300 mg daily for days 1-21) in combination with everolimus (2.5 mg daily for days 1-28) every 28 days. Secondary endpoints were median progression-free survival (mPFS), median overall survival (mOS), response rate, safety, and effect on the retinoblastoma pathway. Results: Twelve patients were enrolled, six at each dose level. Only one patient had a dose-limiting toxicity of a grade 3 rash at the 250 mg dose. The RP2D of ribociclib was 300 mg. mPFS was 1.8 months (95% confidence interval [CI] [0.6-2.1]), and mOS was 3.7 months (95% CI [2.3-5.6]). Two patients (17%) had stable disease at 8 weeks. Pharmacodynamic evaluation demonstrated that CDK4/6-regulated gene expression was significantly decreased on treatment ( n  = 6, p  < 0.001). Conclusion: Ribociclib 300 mg daily for days 1-21 plus everolimus 2.5 mg daily was well tolerated and associated with decreased CDK4/6-regulated gene expression. This combination was not effective as a third-line therapy but does pharmacologically target CDK4/6 in mPC, revealing the potential for benefit in other settings., Competing Interests: B.A.W. receives speaker's bureau honoraria from Lilly, Bayer, Taiho, and Sirtex, and is a consultant for Bayer. M.J.P. is speaker/consultant for AstraZeneca/MedImmune, Caris Life Sciences, Celgene, Halozyme, Merck, Merrimack, RenovoRx, and Sirtex Medical. Travel, accommodations, and expenses support were by AstraZeneca/MedImmune, Caris Life Sciences, Halozyme, Merck, Perthera, and Sirtex Medical. Stock was by Perthera. Research funding to the institution was by ARMO BioSciences, Bavarian Nordic, Bayer, Bristol-Myers Squibb, Calithera Biosciences, Celgene, Celldex, Curegenix, Fibrogen, Genentech, Gilead Sciences, GlaxoSmithKline, Halozyme, Karyopharm Therapeutics, MedImmune, Merck, Novartis, Regeneron, Pfizer, Pharmacyclics, and Tesaro. A.R.H. served as consultant/advisor for Merck, Genentech, BMS, Bayer, Eisai, and AstraZeneca; received honoraria from Eisai, Bayer, BMS, and Exelixis; and research funding from Genentech and Merck Serono. The other authors declare no potential conflicts of interest., (© Benjamin A. Weinberg et al., 2020; Published by Mary Ann Liebert, Inc.)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CDK4/6 inhibitors are now part of the standard armamentarium for hormone receptor-positive breast cancer. In this article, we review the biologic outcomes imposed by these drugs on cancer cells, determinants of response, mechanisms of intrinsic and acquired resistance, as well as combinatorial approaches emanating from mechanistic studies that may allow use of these agents to extend beyond breast cancer. In addition, we will address tumor-, imaging-, and blood-based pharmacodynamic biomarkers that can inform rationally designed trials as clinical development continues.

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.

Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease characterized by the aberrations in multiple genes that drive pathogenesis and limit therapeutic response. While CDK4/6 represents a downstream target of both KRAS mutation and loss of the CDKN2A tumor suppressor in PDAC, clinical and preclinical studies indicate that pharmacological CDK4/6 inhibitors are only modestly effective. Since chemotherapy represents the established backbone of PDAC treatment we evaluated the interaction of CDK4/6 inhibitors with gemcitabine and taxanes that are employed in the treatment of PDAC. Herein, we demonstrate that the difference in mechanisms of actions of chemotherapeutic agents elicit distinct effects on the cellular response to CDK4/6 inhibition. Gemcitabine largely ablates the function of CDK4/6 inhibition in S-phase arrested cells when administered contemporaneously; although, when cells recover from S-phase block they exhibit sensitivity to CDK4/6 inhibition. In contrast, pharmacological inhibition of CDK4/6 yields a cooperative cytostatic effect in combination with docetaxel and prevents adaptation and cell cycle re-entry, which is a common basis for resistance to such agents. Importantly, using organoid and PDX models we could confirm the cooperative effects between chemotherapy and CDK4/6 inhibition in vivo. These data indicate that the combination of cytotoxic and cytostatic agents could represent an important modality in those tumor types that are relatively resistant to CDK4/6 inhibitors.

The p27 protein is a canonical negative regulator of cell proliferation and acts primarily by inhibiting cyclin-dependent kinases (CDKs). Under some circumstances, p27 is associated with active CDK4, but no mechanism for activation has been described. We found that p27, when phosphorylated by tyrosine kinases, allosterically activated CDK4 in complex with cyclin D1 (CDK4-CycD1). Structural and biochemical data revealed that binding of phosphorylated p27 (phosp27) to CDK4 altered the kinase adenosine triphosphate site to promote phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and other substrates. Surprisingly, purified and endogenous phosp27-CDK4-CycD1 complexes were insensitive to the CDK4-targeting drug palbociclib. Palbociclib instead primarily targeted monomeric CDK4 and CDK6 (CDK4/6) in breast tumor cells. Our data characterize phosp27-CDK4-CycD1 as an active Rb kinase that is refractory to clinically relevant CDK4/6 inhibitors., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Genetic alterations are essential for cancer initiation and progression. However, differentiating mutations that drive the tumor phenotype from mutations that do not affect tumor fitness remains a fundamental challenge in cancer biology. To better understand the impact of a given mutation within cancer, RNA-sequencing data was used to categorize mutations based on their allelic expression. For this purpose, we developed the MAXX (Mutation Allelic Expression Extractor) software, which is highly effective at delineating the allelic expression of both single nucleotide variants and small insertions and deletions. Results from MAXX demonstrated that mutations can be separated into three groups based on their expression of the mutant allele, lack of expression from both alleles, or expression of only the wild-type allele. By taking into consideration the allelic expression patterns of genes that are mutated in PDAC, it was possible to increase the sensitivity of widely used driver mutation detection methods, as well as identify subtypes that have prognostic significance and are associated with sensitivity to select classes of therapeutic agents in cell culture. Thus, differentiating mutations based on their mutant allele expression via MAXX represents a means to parse somatic variants in tumor genomes, helping to elucidate a gene's respective role in cancer.

Pancreatic ductal adenocarcinoma (PDAC), like many KRAS-driven tumors, preferentially loses CDKN2A that encodes an endogenous CDK4/6 inhibitor to bypass the RB-mediated cell cycle suppression. Analysis of a panel of patient-derived cell lines and matched xenografts indicated that many pancreatic cancers have intrinsic resistance to CDK4/6 inhibition that is not due to any established mechanism or published biomarker. Rather, there is a KRAS-dependent rapid adaptive response that leads to the upregulation of cyclin proteins, which participate in functional complexes to mediate resistance. In vivo, the degree of response is associated with the suppression of a gene expression signature that is strongly prognostic in pancreatic cancer. Resistance is associated with an adaptive gene expression signature that is common to multiple kinase inhibitors, but is attenuated with MTOR inhibitors. Combination treatment with MTOR and CDK4/6 inhibitors had potent activity across a large number of patient-derived models of PDAC underscoring the potential clinical efficacy.

Recent studies highlight the importance of the RB1 tumor suppressor as a target for cancer therapy. Canonically, RB1 regulates cell cycle progression and represents the downstream target for cyclin-dependent kinase (CDK) 4/6 inhibitors that are in clinical use. However, newly discovered features of the RB1 pathway suggest new therapeutic strategies to counter resistance and improve precision medicine. These therapeutic strategies include deepening cell cycle exit with CDK4/6 inhibitor combinations, selectively targeting tumors that have lost RB1, and expanding therapeutic index by mitigating therapy-associated adverse effects. In addition, RB1 impacts immunological features of tumors and the microenvironment that can enhance sensitivity to immunotherapy. Lastly, RB1 specifies epigenetically determined cell lineage states that are disrupted during therapy resistance and could be re-installed through the direct use of epigenetic therapies. Thus, new opportunities are emerging to improve cancer therapy by exploiting the RB1 pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: Cancer cells often have deficiencies in cell-cycle control mechanisms and could be dependent on specific cell-cycle checkpoints to maintain viability. Because of the documented role of KRAS in driving replication stress, we targeted the checkpoint governing DNA replication using CHK1 kinase inhibitors in pancreatic ductal adenocarcinoma (PDAC) models and examined mechanisms of resistance., Experimental Design: Single-agent efficacy of CHK1 inhibition was investigated in established and primary PDAC lines. Drug screening was performed to identify cooperative agents. In vitro and in vivo studies were employed to interrogate combination treatment efficacy and mechanisms of resistance., Results: Many PDAC models evade single-agent inhibition through mechanisms that allow S-phase progression with CHK1 inhibited. Gene expression analysis revealed FOXM1 as a potential marker of CHK1 sensitivity and defined a form of pancreatic cancer with poor prognosis. Drug screen analysis identified WEE1 as a cooperative agent with CHK1 and was effective in cell culture. In vivo experiments validated the combination efficacy; however, resistance could evolve. Resistance was due to selection of a stable subclone from the original PDX tumor, which harbored high baseline replication stress. In vitro analysis revealed that gemcitabine could eliminate viability in the resistant models. The triplet regimen of gemcitabine, CHK1, and WEE1 inhibition provided strong disease control in all xenograft models interrogated., Conclusions: These results demonstrate the therapeutic resiliency of pancreatic cancer and indicate that coordinately targeting cell-cycle checkpoints in concert with chemotherapy could be particularly efficacious., (©2018 American Association for Cancer Research.)

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent tumorigenic growth, but the role of KRAS in supporting autophagy has not been established. We show that, to our surprise, suppression of KRAS increased autophagic flux, as did pharmacological inhibition of its effector ERK MAPK. Furthermore, we demonstrate that either KRAS suppression or ERK inhibition decreased both glycolytic and mitochondrial functions. We speculated that ERK inhibition might thus enhance PDAC dependence on autophagy, in part by impairing other KRAS- or ERK-driven metabolic processes. Accordingly, we found that the autophagy inhibitor chloroquine and genetic or pharmacologic inhibition of specific autophagy regulators synergistically enhanced the ability of ERK inhibitors to mediate antitumor activity in KRAS-driven PDAC. We conclude that combinations of pharmacologic inhibitors that concurrently block both ERK MAPK and autophagic processes that are upregulated in response to ERK inhibition may be effective treatments for PDAC.

Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Motivation: Tumor genome sequencing offers great promise for guiding research and therapy, but spurious variant calls can arise from multiple sources. Mouse contamination can generate many spurious calls when sequencing patient-derived xenografts. Paralogous genome sequences can also generate spurious calls when sequencing any tumor. We developed a BLAST-based algorithm, Mouse And Paralog EXterminator (MAPEX), to identify and filter out spurious calls from both these sources., Results: When calling variants from xenografts, MAPEX has similar sensitivity and specificity to more complex algorithms. When applied to any tumor, MAPEX also automatically flags calls that potentially arise from paralogous sequences. Our implementation, mapexr, runs quickly and easily on a desktop computer. MAPEX is thus a useful addition to almost any pipeline for calling genetic variants in tumors., Availability and Implementation: The mapexr package for R is available at https://github.com/bmannakee/mapexr under the MIT license., Contact: mannakee@email.arizona.edu or rgutenk@email.arizona.edu or eknudsen@email.arizona.edu., Supplementary Information: Supplementary data are available at Bioinformatics online.

Objective: Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease with the worst survival rate of common solid tumours. Preclinical models that accurately reflect the genetic and biological diversity of PDAC will be important for delineating features of tumour biology and therapeutic vulnerabilities., Design: 27 primary PDAC tumours were employed for genetic analysis and development of tumour models. Tumour tissue was used for derivation of xenografts and cell lines. Exome sequencing was performed on the originating tumour and developed models. RNA sequencing, histological and functional analyses were employed to determine the relationship of the patient-derived models to clinical presentation of PDAC., Results: The cohort employed captured the genetic diversity of PDAC. From most cases, both cell lines and xenograft models were developed. Exome sequencing confirmed preservation of the primary tumour mutations in developed cell lines, which remained stable with extended passaging. The level of genetic conservation in the cell lines was comparable to that observed with patient-derived xenograft (PDX) models. Unlike historically established PDAC cancer cell lines, patient-derived models recapitulated the histological architecture of the primary tumour and exhibited metastatic spread similar to that observed clinically. Detailed genetic analyses of tumours and derived models revealed features of ex vivo evolution and the clonal architecture of PDAC. Functional analysis was used to elucidate therapeutic vulnerabilities of relevance to treatment of PDAC., Conclusions: These data illustrate that with the appropriate methods it is possible to develop cell lines that maintain genetic features of PDAC. Such models serve as important substrates for analysing the significance of genetic variants and create a unique biorepository of annotated cell lines and xenografts that were established simultaneously from same primary tumour. These models can be used to infer genetic and empirically determined therapeutic sensitivities that would be germane to the patient., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Cancer biology is influenced by the tumor microenvironment, which impacts disease prognosis and therapeutic interventions. The inter-relationship of tumor-infiltrating lymphocytes, immune response regulators, and a glycolytic tumor environment was evaluated in a cohort of 183 largely consecutive patients with triple negative breast cancer diagnosis. High levels of tumor-infiltrating lymphocytes were associated with improved survival of triple negative breast cancer cases. However, elevated levels of PD-L1, CD163, and FOXP3 were individually associated with significantly decreased overall survival. These three determinants were significantly correlated, and could serve to differentiate the prognostic significance of tumor-infiltrating lymphocytes. Interestingly, a glycolytic tumor environment, as determined by the expression of MCT4 in the tumor stroma, was associated with the immune evasive environment and poor prognosis. Clustering of all markers defined four distinct triple negative breast cancer subtypes that harbored prognostic significance in multivariate analysis. Immune and metabolic markers stratified triple negative breast cancer into subtypes that have prognostic significance and implications for therapies targeting immune checkpoints and tumor metabolism.