CDK2 inhibition disorders centrosome stoichiometry and alters cellular outcomes in aneuploid cancer cells.

Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of aneuploid cancers. This study elucidated how CDK2 antagonism affected centrosome stoichiometry. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent imaging were used. Studies interrogated multipolar mitosis after pharmacologic or genetic repression of CDK2. CDK2/9 antagonism with CYC065 (Fadraciclib)-treatment disordered centrosome stoichiometry in aneuploid cancer cells, preventing centrosome clustering. This caused ring-like chromosomes or multipolar cancer cells to form before onset of cell death. Intriguingly, CDK2 inhibition caused a statistically significant increase in single centrioles rather than intact centrosomes with two centrioles in cancer cells having chromosome rings or multipolarity. Statistically significant alterations in centrosome stoichiometry were undetected in other mitotic cancer cells. To confirm this pharmacodynamic effect, CDK2 but not CDK9 siRNA-mediated knockdown augmented cancer cells with chromosome ring or multipolarity formation. Notably, engineered gain of CDK2, but not CDK9 expression, reversed emergence of cancer cells with chromosome rings or multipolarity, despite CYC065-treatment. In marked contrast, CDK2 inhibition of primary human alveolar epithelial cells did not confer statistically significant increases of cells with ring-like chromosomes or multipolarity. Hence, CDK2 antagonism caused differential effects in malignant versus normal alveolar epithelial cells. Translational relevance was confirmed by CYC065-treatment of syngeneic lung cancers in mice. Mitotic figures in tumors exhibited chromosome rings or multipolarity. Thus, CDK2 inhibition preferentially disorders centrosome stoichiometry in cancer cells. Engaging this disruption is a strategy to explore against aneuploid cancers in future clinical trials.

A novel retinoic acid receptor-γ agonist antagonizes immune checkpoint resistance in lung cancers by altering the tumor immune microenvironment.

All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA's anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA's anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice-a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.

NOXA expression is downregulated in human breast cancer undergoing incomplete pathological response and senescence after neoadjuvant chemotherapy.

Neoadjuvant chemotherapy (NAC) is a frequently utilized approach to treat locally advanced breast cancer, but, unfortunately, a subset of tumors fails to undergo complete pathological response. Apoptosis and therapy-induced senescence (TIS) are both cell stress mechanisms but their exact role in mediating the pathological response to NAC is not fully elucidated. We investigated the change in expression of PAMIP1, the gene encoding for the pro-apoptotic protein, NOXA, following NAC in two breast cancer gene datasets, and the change in NOXA protein expression in response to NAC in 55 matched patient samples (pre- and post-NAC). PAMIP1 expression significantly declined in post-NAC in the two sets, and in our cohort, 75% of the samples exhibited a downregulation in NOXA post-NAC. Matched samples that showed a decline in NOXA post-NAC were examined for TIS based on a signature of downregulated expression of Lamin-B1 and Ki-67 and increased p16INK4a, and the majority exhibited a decrease in Lamin B1 (66%) and Ki-67 (80%), and increased p16INK4a (49%). Since our cohort consisted of patients that did not develop complete pathological response, such findings have clinical implications on the role of TIS and NOXA downregulation in mediating suboptimal responses to the currently established NAC.

The BET inhibitor/degrader ARV-825 prolongs the growth arrest response to Fulvestrant + Palbociclib and suppresses proliferative recovery in ER-positive breast cancer.

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.

Autophagy-Dependent Sensitization of Triple-Negative Breast Cancer Models to Topoisomerase II Poisons by Inhibition of the Nucleosome Remodeling Factor.

Epigenetic regulators can modulate the effects of cancer therapeutics. To further these observations, we discovered that the bromodomain PHD finger transcription factor subunit (BPTF) of the nucleosome remodeling factor (NURF) promotes resistance to doxorubicin, etoposide, and paclitaxel in the 4T1 breast tumor cell line. BPTF functions in promoting resistance to doxorubicin and etoposide, but not paclitaxel, and may be selective to cancer cells, as a similar effect was not observed in embryonic stem cells. Sensitization to doxorubicin and etoposide with BPTF knockdown (KD) was associated with increased DNA damage, topoisomerase II (TOP2) crosslinking and autophagy; however, there was only a modest increase in apoptosis and no increase in senescence. Sensitization to doxorubicin was confirmed in vivo with the syngeneic 4T1 breast tumor model using both genetic and pharmacologic inhibition of BPTF. The effects of BPTF inhibition in vivo are autophagy dependent, based on genetic autophagy inhibition. Finally, treatment of 4T1, 66cl4, 4T07, MDA-MB-231, but not ER-positive 67NR and MCF7 breast cancer cells with the selective BPTF bromodomain inhibitor, AU1, recapitulates genetic BPTF inhibition, including in vitro sensitization to doxorubicin, increased TOP2-DNA crosslinks and DNA damage. Taken together, these studies demonstrate that BPTF provides resistance to the antitumor activity of TOP2 poisons, preventing the resolution of TOP2 crosslinking and associated autophagy. These studies suggest that BPTF can be targeted with small-molecule inhibitors to enhance the effectiveness of TOP2-targeted cancer chemotherapeutic drugs. IMPLICATIONS: These studies suggest NURF can be inhibited pharmacologically as a viable strategy to improve chemotherapy effectiveness.

A Novel CDK2/9 Inhibitor CYC065 Causes Anaphase Catastrophe and Represses Proliferation, Tumorigenesis, and Metastasis in Aneuploid Cancers.

Cyclin-dependent kinase 2 (CDK2) antagonism inhibits clustering of excessive centrosomes at mitosis, causing multipolar cell division and apoptotic death. This is called anaphase catastrophe. To establish induced anaphase catastrophe as a clinically tractable antineoplastic mechanism, induced anaphase catastrophe was explored in different aneuploid cancers after treatment with CYC065 (Cyclacel), a CDK2/9 inhibitor. Antineoplastic activity was studied in preclinical models. CYC065 treatment augmented anaphase catastrophe in diverse cancers including lymphoma, lung, colon, and pancreatic cancers, despite KRAS oncoprotein expression. Anaphase catastrophe was a broadly active antineoplastic mechanism. Reverse phase protein arrays (RPPAs) revealed that along with known CDK2/9 targets, focal adhesion kinase and Src phosphorylation that regulate metastasis were each repressed by CYC065 treatment. Intriguingly, CYC065 treatment decreased lung cancer metastases in in vivo murine models. CYC065 treatment also significantly reduced the rate of lung cancer growth in syngeneic murine and patient-derived xenograft (PDX) models independent of KRAS oncoprotein expression. Immunohistochemistry analysis of CYC065-treated lung cancer PDX models confirmed repression of proteins highlighted by RPPAs, implicating them as indicators of CYC065 antitumor response. Phospho-histone H3 staining detected anaphase catastrophe in CYC065-treated PDXs. Thus, induced anaphase catastrophe after CYC065 treatment can combat aneuploid cancers despite KRAS oncoprotein expression. These findings should guide future trials of this novel CDK2/9 inhibitor in the cancer clinic.

Clearance of therapy-induced senescent tumor cells by the senolytic ABT-263 via interference with BCL-XL -BAX interaction.

Tumor cells undergo senescence in response to both conventional and targeted cancer therapies. The induction of senescence in response to cancer therapy can contribute to unfavorable patient outcomes, potentially including disease relapse. This possibiliy is supported by our findings that tumor cells induced into senescence by doxorubicin or etoposide can give rise to viable tumors in vivo. We further demonstrate sensitivity of these senescent tumor cells to the senolytic ABT-263 (navitoclax), therefore providing a "two-hit" approach to eliminate senescent tumor cells that persist after exposure to chemotherapy or radiation. The sequential combination of therapy-induced senescence and ABT-263 could shift the response to therapy toward apoptosis by interfering with the interaction between BCL-XL and BAX. The administration of ABT-263 after either etoposide or doxorubicin also resulted in marked, prolonged tumor suppression in tumor-bearing animals. These findings support the premise that senolytic therapy following conventional cancer therapy may improve therapeutic outcomes and delay disease recurrence.

Studies of Non-Protective Autophagy Provide Evidence that Recovery from Therapy-Induced Senescence is Independent of Early Autophagy.

Autophagy and senescence, predominant responses that may dictate cell fate after chemotherapy or radiation, often occur in tandem. Cells in states of senescence and/or autophagy are frequently growth arrested. We have previously reported that tumor cells induced into senescence by therapy can re-emerge from the growth-arrested state, a phenomenon termed proliferative recovery. The current work shows that, while tumor cells collaterally induced into senescence and autophagy by etoposide, doxorubicin, or radiation undergo proliferative recovery, neither pharmacological nor genetic inhibition of early autophagy alter the extent of senescence or the ability of cells to recover from senescence. These findings confirm and extend our previous observations, essentially dissociating senescence from autophagy, and further indicate that re-emergence from senescence does not appear to be facilitated by or dependent on autophagy. Our results also provide additional evidence for the promotion of the non-protective form of autophagy by both chemotherapeutic drugs and radiation, which may complicate current efforts to inhibit autophagy for therapeutic benefit.

Roles of autophagy in breast cancer treatment: Target, bystander or benefactor.

Despite the availability of largely effective treatments for breast cancer, such as the combination of aromatase inhibitors or anti-estrogens with cdk4/6 inhibitors for estrogen receptor positive/Her 2 negative breast cancer, breast cancer remains a significant cause of morbidity and mortality. This is due, in large part, to a very limited understanding of the mechanisms underlying the failure of conventional therapies and disease recurrence after tumor dormancy. One cellular process that is activated in response to the majority of breast cancer treatments is autophagy. Proven to be an indispensable cellular function, autophagy is largely accepted as a pro-survival mechanism in tumor cells and has consequently generated significant interest in cancer research and treatment strategies. Autophagy plays multiple and often disparate roles during different stages of tumorigenesis and in response to anti-tumor treatments; in fact, autophagy is induced by almost all conventional treatments of breast cancer and is considered a target for pharmacologic blockade in the clinic. Consequently, it is important to further our understanding of this process and its role in breast cancer.

Tumor Cell Escape from Therapy-Induced Senescence as a Model of Disease Recurrence after Dormancy.

Senescence, a durable form of growth arrest, represents a primary response to numerous anticancer therapies. Although the paradigm that senescence is "irreversible" has largely withstood the findings of tumor cell recovery from what has been termed "pseudo-senescence" or "senescence-like arrest," a review of the literature suggests that therapy-induced senescence in tumor cells is not obligatorily a permanent cell fate. Consequently, we propose that senescence represents one avenue whereby tumor cells evade the direct cytotoxic impact of therapy, thereby allowing for prolonged survival in a dormant state, with the potential to recover self-renewal capacity and contribute to disease recurrence.

Tumor cell escape from therapy-induced senescence.

H460 non-small cell lung, HCT116 colon and 4T1 breast tumor cell lines induced into senescence by exposure to either etoposide or doxorubicin were able to recover proliferative capacity both in mass culture and when enriched for the senescence-like phenotype by flow cytometry (based on ß-galactosidase staining and cell size, and a senescence-associated reporter, BTG1-RFP). Recovery was further established using both real-time microscopy and High-Speed Live-Cell Interferometry (HSLCI) and was shown to be accompanied by the attenuation of the Senescence-Associated Secretory Phenotype (SASP). Cells enriched for the senescence-like phenotype were also capable of forming tumors when implanted in both immunodeficient and immunocompetent mice. As chemotherapy-induced senescence has been identified in patient tumors, our results suggest that certain senescence-like phenotypes may not reflect a terminal state of growth arrest, as cells that recover with self-renewal capacity may ultimately contribute to disease recurrence.

Autophagy-deficient breast cancer shows early tumor recurrence and escape from dormancy.

Breast cancer patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu-overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting response to immunotherapy. We explored two strategies: 1) transient blockade of autophagy with chloroquine (CQ), which blocks fusion of autophagosomes and lysosomes during ADR treatment, and 2) permanent inhibition of autophagy by a stable knockdown of ATG5 (ATG5KD), which inhibits the formation of autophagosomes in MMC during and after ADR treatment. We found that while CQ prolonged tumor dormancy, but that stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, ATG5KD MMC contained an increased frequency of ADR-induced polyploid-like cells and rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell-intrinsic autophagy delays tumor relapse, in part, by inhibiting the formation of polyploid-like tumor dormancy.

Leveraging Epigenetics to Enhance the Cellular Response to Chemotherapies and Improve Tumor Immunogenicity.

Cancer chemotherapeutic drugs have greatly advanced our ability to successfully treat a variety of human malignancies. The different forms of stress produced by these agents in cancer cells result in both cell autonomous and cell nonautonomous effects. Desirable cell autonomous effects include reduced proliferative potential, cellular senescence, and cell death. More recently recognized cell nonautonomous effects, usually in the form of stimulating an antitumor immune response, have significant roles in therapeutic efficiency for a select number of chemotherapies. Unfortunately, the success of these therapeutics is not universal as not all tumors respond to treatment, and those that do respond will frequently relapse into therapy-resistant disease. Numerous strategies have been developed to sensitize tumors toward chemotherapies as a means to either improve initial responses, or serve as a secondary treatment strategy for therapy-resistant disease. Recently, targeting epigenetic regulators has emerged as a viable method of sensitizing tumors to the effects of chemotherapies, many of which are cytotoxic. In this review, we summarize these strategies and propose a path for future progress.

The potentially conflicting cell autonomous and cell non-autonomous functions of autophagy in mediating tumor response to cancer therapy.

Autophagy, a virtually uniform response to external stress such as that induced by chemotherapy and radiation, is generally considered to be cytoprotective in function, providing a foundation for multiple clinical trials designed to enhance therapeutic response via autophagy inhibition. However, this cell autonomous response can also be cytotoxic or nonprotective, with the consequence that autophagy inhibition would be counterproductive or ineffective, respectively. The non-cell autonomous function of autophagy remains quite controversial, with evidence both for and against autophagy-mediated activation of the immune system. If autophagy inhibition antagonizes the immune response, this would likely interfere with the potential sensitization resulting from suppression of the cell autonomous protective function. An additional complication, which has rarely been considered, is the nature of the contribution of therapy-induced autophagy in the tumor microenvironment, particularly the tumor stroma. Taken together, it is likely that the outcome of the current clinical trials, whether positive or negative, will be difficult to interpret given the complexity of the role of autophagy relating to the tumor cell (cell autonomous), the immune system (cell non-autonomous) and the tumor microenvironment.