Aneuploidy is a cancer hallmark that causes resistance to anticancer drugs and promotes aggressive tumors. Thus, aneuploidy is a consequential feature of human malignancy. This review addresses how cyclin-dependent kinase 2 (CDK2) inhibition targets a broad array of aneuploid cancers for proapoptotic death by engaging a death program called anaphase catastrophe. This program eliminates aneuploid cancers while sparing non-aneuploid epithelial cells, thereby providing a favorable therapeutic window. Despite CDK2 inhibition, a residual population of polyploid cancer cells persists in both in vitro and in vivo settings. This polyploid cancer cell population is resistant to apoptosis conferred by CDK2 inhibition of aneuploid cancers. This provides a basis for clinical drug resistance. The triggering apoptotic death of aneuploid cancers via CDK2 antagonism is compromised by the presence of this apoptosis-resistant population. To elucidate the nature of this polyploid population, these apoptotic-resistant cells were isolated and were found enriched for expressed cyclin-dependent kinase 1 (CDK1) and kinesin superfamily proteins (KIFs). Intriguingly, combining CDK2 inhibition with antagonists for CDK1 or KIF species markedly promoted anticancer effects in aneuploid cancers. This clinically-tractable combined regimen is hypothesized to expose aneuploid cancers to eradication after CDK2 antagonism. Future clinical trials should explore this possibility.

Ubiquitin-Specific Protease 18 (USP18) is a deISGylation enzyme and antineoplastic target. To develop USP18 inhibitors, an enzymatically active human recombinant USP18 protein was engineered suitable for high-throughput screening of ~80,000 chemical compounds. Three of them substantially inhibited USP18 enzymatic activity, with β-lapachone having prominent antineoplastic activity. Independent β-lapachone treatments of murine and human lung cancer cell lines statistically significantly reduced proliferation and increased apoptosis. Gain of USP18 expression antagonized these effects. β-Lapachone treatments statistically significantly repressed lung cancer xenograft growth. β-Lapachone increased reactive oxygen species (ROS), but antineoplastic effects occurred at dosages with negligible ROS production. ROS scavenger treatments did not rescue β-lapachone effects at these concentrations, consistent with an ROS-independent mechanism. IFN-Stimulated Response Element (ISRE) reporter assays following β-lapachone treatment activated this reporter. USP18 cotransfection antagonized this activity. β-Lapachone treatments increased global ISGylation. RNA-seq of lung cancer cells engineered with or without enhanced USP18 expression showed specific pathways affected by β-lapachone treatment. Proteomic analysis of these treated cells revealed known and new ISGylated proteins. In silico modeling identified a unique USP18 pocket where these USP18 inhibitors bind. Engineered mutation of this pocket disrupted β-lapachone activity. Taken together, β-lapachone is an antineoplastic tool compound useful for USP18 inhibitor development.

Supernumerary centrosomes are a hallmark of cancer. To maintain viability, cancer cells cluster these centrosomes during mitosis, enabling bipolar division similar to that of normal cells. Disruption of this centrosome clustering leads to multipolar anaphase and apoptosis (anaphase catastrophe), which selectively eliminates cancer cells harboring supernumerary centrosomes. In this context, because the motor protein KIFC1 contributes to centrosome clustering, we investigated whether targeting of this mechanism through KIFC1 inhibition could be exploited in small-cell lung cancer (SCLC), an aggressive malignancy with limited treatment options and poor prognosis. Through in silico and in vitro analyses, as well as IHC of clinical samples, we found that KIFC1 is overexpressed and that centrosome amplification occurs more frequently in SCLC compared with normal tissues and other cancer types. Pharmacological and genetic inhibition of KIFC1 disrupted the clustering of supernumerary centrosomes, triggered multipolar mitosis, and exerted antineoplastic effects in SCLC cells, with minimal effects on noncancerous cells. These findings were validated and extended in vivo using SCLC xenograft models. Finally, cotargeting KIFC1 and the centrosome duplication regulator PLK4 further enhanced growth suppression in SCLC cells. Together, these results suggest that disrupting centrosome clustering and triggering anaphase catastrophe via KIFC1 inhibition may represent a promising therapeutic strategy for SCLC.

Abstract Cancer cells often possess more than two centrosomes, which is one of the hallmarks of cancer. By clustering these supernumerary centrosomes during mitosis, cancer cells can achieve bipolar division. We previously reported that in non-small cell lung cancer, CDK2 inhibition suppresses centrosome clustering, leading to multipolar cell division and apoptotic cell death—a phenomenon termed anaphase catastrophe. Because normal cells have only two centrosomes, anaphase catastrophe does not appreciably affect them, making it a promising therapeutic strategy for selectively eliminating cancer cells. However, as CDK2 inhibition also interferes with normal cell cycle progression, identifying therapeutic targets that can induce anaphase catastrophe preferentially is desirable. Motor protein KIFC1 has been implicated in centrosome clustering. Here, we investigated its potential as a novel therapeutic target to induce anaphase catastrophe in small cell lung cancer (SCLC). Owing to p53 inactivation, SCLC is expected to frequently harbor supernumerary centrosomes and be vulnerable to mitotic abnormalities, including anaphase catastrophe. First, in vitro experiments using multiple SCLC cell lines were conducted. Immunofluorescence staining for pericentrin confirmed that centrosome amplification occurs more frequently in SCLC cells than in normal human bronchial epithelial (NHBE) cells. Western blotting revealed that KIFC1 was overexpressed in SCLC cells as compared with NHBE cells. Functional analyses, both pharmacologic (using a specific inhibitor) and genetic (using siRNAs and the CRISPR-Cas9 system), demonstrated that inhibition of KIFC1 induced apoptosis and suppressed proliferation in SCLC cell lines, whereas these effects were largely absent in NHBE cells. Furthermore, immunostaining for α-tubulin, pericentrin, and DAPI showed a statistically significant increase in multipolar mitotic cells and a decrease in cells with clustered supernumerary centrosomes among dividing cells, implicating KIFC1 inhibition in conferring anaphase catastrophe in SCLC. These findings were validated and extended in the in vivo setting. In human SCLC cell-derived xenograft models, administration of KIFC1 inhibitor statistically significantly suppressed tumor growth. Staining of excised tumors for pericentrin revealed reduced numbers of cells with clustered supernumerary centrosomes and increased numbers undergoing multipolar mitosis, consistent with in vitro findings and indicative of anaphase catastrophe induction. Finally, potential combination therapies with other pharmacologic targets were explored and will be presented. In conclusion, inhibition of KIFC1 disrupts the clustering of supernumerary centrosomes and caused anaphase catastrophe, indicating its promise as a novel therapeutic target for SCLC. Citation Format: Minemichi Toda, Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Takahiro Iida, Hiroaki Ikushima, Takahiro Ando, Akiko Kunita, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. Targeting KIFC1 induces anaphase catastrophe in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5687.

Aneuploidy, a cancer hallmark, drives chromosomal instability, drug resistance, and clinically aggressive tumors. Cyclin-dependent kinase 2 (CDK2) antagonism with independent inhibitors or CDK2 knockdown triggered anaphase catastrophe. This disrupts supernumerary centrosome clustering, causing multipolar division and apoptosis. Time-lapse fluorescence microscopy of fluorescent ubiquitination-based cell cycle indicator (FUCCI) cell cycle probes transduced into aneuploid lung cancer cells revealed distinct fates of bipolar and polyploid cells after CDK2 inhibition. Apoptosis occurred in multipolar progeny but was repressed in persistent polyploid cancer cells. RNA-Seq analyses after CDK2 inhibition of 4N versus 2N lung cancer cells were enriched for CDK1 pathway and KIF family members. The Cancer Genome Atlas (TCGA) analysis of lung cancers indicated that CDK1 and KIF family member overexpression was associated with an unfavorable survival. Intravital microscopy of transplanted lung cancer cells in mice extended findings from the in vitro to in vivo settings. CDK2 inhibition of tumor-bearing mice produced polyploid cancer cells in vivo. These cancer cells were resistant to apoptosis and proliferated despite CDK2 inhibition. In contrast, polyploid populations were rarely detected in CDK2-inhibited human alveolar epithelial cells. These findings are translationally relevant. Combined targeting of CDK2 with CDK1 or kinesin family member antagonists should eliminate polyploid cancer cells, promote apoptosis, and augment antineoplastic effects.

Cancer cells often have more than two centrosomes (centrosome amplification). This is a cancer hallmark. Cancer cells cluster supernumerary centrosomes during mitosis so that they can undergo bipolar mitosis, similar to normal cells. We previously reported that inhibition of centrosome clustering by CDK2 antagonism in non-small lung cell cancer caused multipolar cell division and apoptotic death, termed anaphase catastrophe. Anaphase catastrophe preferentially affects cancer cells, sparing normal cells with two centrosomes. This therapeutic window implicates triggering anaphase catastrophe as a promising antineoplastic pathway. Motor protein X is reported to affect centrosome clustering but is not essential in normal cells. Small cell lung cancer (SCLC) has p53 inactivation and a poor prognosis with limited therapeutic options. Loss of p53 function disrupts the cell cycle checkpoint and is associated with centrosome amplification. We hypothesized that SCLC is vulnerable to mitotic abnormalities, including anaphase catastrophe. We explore here protein X as a novel therapeutic target that triggers anaphase catastrophe and apoptosis in SCLC. In vitro studies of multiple SCLC cell lines were performed. Immunofluorescence staining of pericentrin revealed higher frequency of centrosome amplification in SCLC than in normal human bronchial epithelial (NHBE) cells. Immunoblotting indicated that motor protein X was over-expressed in SCLC cells compared to NHBE cells. Functional analyses of protein X was done using siRNAs, CRISPR-Cas9 system, and a specific inhibitor. Protein X inhibition reduced cell proliferation and triggered apoptosis in the examined SCLC cell lines. In NHBE cells with infrequent centrosome amplification, these effects were rarely observed. To elucidate the mechanism for these antineoplastic effects, the mitotic status of SCLC cells was studied by staining for α-tubulin, pericentrin, and DAPI. Cells with clustered supernumerary centrosomes were statistically significantly decreased, while the number of multipolar mitotic cells were statistically significantly increased upon protein X inhibition. This indicated that targeting the protein X produced anaphase catastrophe in SCLC cells. We confirmed and extended these in vitro findings to the in vivo setting. The protein X inhibitor statistically significantly suppressed the growth of NCI-H146 tumor xenografts in mice. Pericentrin staining of resected treated xenografts revealed that the protein X inhibitor antagonized clustering of supernumerary centrosomes and led to multipolar mitosis in vivo. Taken together, prevention of supernumerary centrosome clustering by targeting the motor protein X is an attractive therapeutic strategy for SCLC. Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Takahiro Iida, Takahiro Ando, Akiko Kunita, Kousuke Watanabe, Yasunori Ohta, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. Prevention of centrosome clustering by targeting the motor protein X: A promising therapeutic strategy for small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 362.

Aneuploidy, a hallmark of cancer, drives chromosomal instability and antineoplastic drug resistance. Common in hematologic and solid tumors, centrosome amplification leads to supernumerary centrosomes, causing chromosomal missegregation in dividing cancer cells. This imbalance produces progeny with abnormal chromosome counts, leading to cell death, resistance to antineoplastics, and clinically-aggressive tumors. This study examined the effects of CDK2 inhibition on aneuploid lung cancer cells and tumors. It revealed that CDK2 antagonism triggers anaphase catastrophe through centrosome clustering disruption and multipolar mitosis. Unexpectedly, this also leads to the formation of a persistent population of polyploid, apoptosis-resistant cells. Using independent CDK2 inhibitors and shRNAs, we observed that some cancer cells underwent multipolar division and subsequent cell death. Others exhibited cytokinesis failure, resulting in multinucleated, and polyploid cancer cells that continued to survive and proliferate despite continuous CDK2 inhibition. Through time-lapse imaging with transfected FUCCI cell cycle probes and RNA-seq analysis of polyploid versus diploid populations, we identified upregulated CDK1-related pathways and kinesin (KIF) family members in the polyploid cellular population. This likely contributes to the persistent polyploid population, despite CDK2 antagonism. These findings were independently validated in vivo using intravital microscopy and patient-derived xenograft (PDX) lung cancer models. Intriguingly, polyploid and multinucleated cancer cells were found to proliferate post-CDK2 inhibitor treatments. In marked contrast, a polyploid population was not found in CDK2 inhibited primary human alveolar epithelial cells, indicating that this is a pathway that is preferentially activated in aneuploid cancer cells. Analysis of gene expression in TCGA lung cancer datasets linked CDK1 and KIF family overexpression to an unfavorable patient survival, implicating this persistent polyploid population as contributing to poor survival outcomes in lung cancer. These findings are relevant to the clinical development of CDK2 inhibitors. They indicate that despite the pro-apoptotic effects of CDK2 antagonism in aneuploid lung cancer cells, a population of drug-resistant, polyploid cells persists. We propose that a combinatorial therapy targeting CDK2 alongside polo-like kinase 4 (PLK4), CDK1 or kinesin family members would further disrupt centrosome clustering, eliminate polyploid cancer cells, and enhance tumor cell apoptosis and antineoplastic effects. This dual approach would enhance the anti-tumor efficacy of CDK2 inhibition, potentially overcoming resistance mechanisms. This provides a promising strategy to eradicate persistent lung cancer cells following CDK2 inhibition. Liliya Tyutyunyk-Massey, Zibo Chen, Xiuxia Liu, Masanori Kawakami, Adam Harned, Yeap Ng, Brian Luke, Sameul C. Okpechi, Blessing Ogunlade, Yair Alfaro, Roberto Weigert, Kedar Narayan, Xi Liu, Ethan Dmitrovsky. CDK2 inhibition produces persistent polyploid cancer cells with a survival advantage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5327.

Abstract Genomic instability is a hallmark of cancer. Unlike normal diploid cells, cancer cells exhibit abnormal centrosome numbers and chromosomal instability. Mitosis with supernumerary centrosomes yields progeny with aberrant chromosome segregation and aneuploidy. While most progeny cells with abnormal chromosomes are eliminated through apoptosis, some have distinct fates with a growth advantage. These cancer cells can acquire resistance to antineoplastic agents. This confers an aggressive tumor biology and unfavorable clinical outcomes. Cyclin-dependent kinase 2 (CDK2) regulates cell cycle progression and the centrosome cycle. We previously reported that CDK2 inhibition prevents supernumerary centrosome clustering, causing multipolar mitosis and anaphase catastrophe in lung and other aneuploid cancers. This was associated with residual transplanted tumors in mice. To elucidate cell fates of progeny with multipolar mitosis after CDK2/9 inhibition with CYC065 (0.2 µM) treatment, a panel of murine and human lung cancer cells underwent time-lapse fluorescent microscopy. This elucidated multipolar mitotic events within the progeny. Intriguingly, distinct outcomes occurred after mitosis including formation of multipolar and multinucleated cells. Cell death of progeny is the predominant fate, but some progeny survive despite continuous CDK2 inhibition. Surprisingly, some of these cells fuse together. Those multinucleated cells can undergo multiple cell cycles without successful cytokinesis. To confirm these effects were through CDK2 inhibition, the selective CDK2 inhibitor Tagtociclib (PF-07104091, at the 2 µM dosage) was used. This treatment statistically-significantly increased multipolar lung cancer cells and increased apoptotic death during time-lapse fluorescent microscopy. Findings were independently validated by CDK2 shRNA knockdown. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent staining revealed the ultrastructure of multinucleated cells. Insights into tumor biology came from intravital imaging of transplanted lung cancer cells in mice. Multipolar and multinuclear cells followed CDK2 antagonism. Incucyte® Live-Cell Analysis System and Artificial Intelligence-based imaging determined distinct growth responses in aneuploid as compared to non-aneuploid lung cancer cells after CDK2 inhibitor treatments. In summary, these findings are translationally relevant. CDK2 inhibition of aneuploid lung cancers yields distinct cell fates. This is linked to resistance to CDK2 antagonism and to formation of residual in vivo tumors. Citation Format: Liliya Tyutyunyk, Zibo Chen, Xiuxia Liu, Yeap Ng, Aayush Bhatawadekar, Kedar Narayan, Roberto Weigert, Xi Liu, Ethan Dmitrovsky. Elucidation of the fates of CDK2 inhibited aneuploid and residual lung cancers [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 2117.

Abstract Cancer cells often have more than two centrosomes (supernumerary centrosomes) and this is one of the hallmarks of cancer. We previously demonstrated that CDK2 antagonism inhibits clustering of supernumerary centrosomes at mitosis leading to multipolar cell division and apoptotic death in non-small cell lung cancer. This antineoplastic process was designated anaphase catastrophe. Anaphase catastrophe can preferentially affect cancer cells sparing normal cells with two centrosomes. On the other hand, as CDK2 functions in normal cell cycle progression, its inhibition can also affect normal and malignant cells. Identification of a novel drug target that would preferentially cause anaphase catastrophe is needed. The motor protein KIFC1 affects centrosome clustering. Small cell lung cancer (SCLC), known for its poor prognosis with limited therapeutic options, displays disruptions in the cell cycle checkpoint mechanism due to inactivation of p53 and RB. We hypothesized SCLC was susceptible to mitotic abnormalities, including anaphase catastrophe. In this study, we explored the possibility of KIFC1 as a novel therapeutic target that triggers anaphase catastrophe in SCLC. In-silico GEO database analysis revealed elevated levels of KIFC1 and PLK4 in SCLCs compared with other cancers and normal tissues. PLK4 plays a key role in centrosome amplification. Our in-silico analysis results imply that centrosome amplification and their clustering frequently occur in SCLC. We performed in-vitro functional analysis of KIFC1 inhibition in SCLC cell lines (NCI-H146, SHP77, NCI-H209, and NCI-H524 cells), using siRNAs and CRISPR-Cas9 system for genetic inhibition and AZ82, a specific KIFC1 inhibitor, for pharmacologic inhibition of KIFC1. Cell proliferation was measured by the CellTiter-Glo Luminescent Assay and apoptosis was scored by annexin V and PI staining followed by flow cytometry analysis. Cell growth was reduced and apoptosis was statistically significantly increased after genetic inhibition of KIFC1 by siRNAs and by use of the CRISPR-Cas9 system in SCLC cell lines. These antineoplastic effects also occurred after AZ82 treatments, in a dose-dependent manner in these SCLC cell lines. To examine the mitotic status of SCLC cells after KIFC1 inhibition, SCLC cells were stained with α-tubulin antibody and DAPI before analysis with a fluorescence microscope. Besides normal bipolar mitotic cells, multipolar mitotic cells were observed, and their population was increased after KIFC1 knockdown with siRNAs. This indicated that clustering of supernumerary centrosomes was inhibited by targeting KIFC1 in SCLC cells. Taken together, KIFC1 is a potential therapeutic target to induce anaphase catastrophe in cancer cells. Targeting KIFC1 is a novel therapeutic strategy to consider for SCLC that currently has limited treatment options. Citation Format: Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Yuriko Sugiura, Takahiro Iida, Takahiro Ando, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe [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 3347.

Background: The consequence of diabetes on lung cancer overall survival (OS) is debated. This retrospective study used 2 large lung cancer databases to assess comprehensively diabetes effects on lung cancer OS in diverse demographic populations, including health disparity. Methods: The University of Texas MD Anderson Cancer Center database (32 643 lung cancer patients with 11 973 patients with diabetes) was extracted from electronic health records (EHRs) using natural language processing (NLP). Associations were between diabetes and lung cancer prognostic features (age, sex, race, body mass index [BMI], insurance status, smoking, stage, and histopathology). Hemoglobin A1C (HgbA1c) and glucose levels assessed glycemic control. Validation was with a Louisiana cohort (17 768 lung cancer patients with 5402 patients with diabetes) enriched for health disparity cases. Kaplan-Meier analysis, log-rank test, multivariable Cox proportional hazard models, and survival tree analyses were employed. Results: Lung cancer patients with diabetes exhibited marginally elevated OS or no statistically significant difference versus nondiabetic patients. When examining OS for 2 glycemic levels (HgbA1c > 7.0 or glucose > 154 mg/dL vs HgbA1c > 9.0 or glucose > 215 mg/dL), a statistically significant improvement in OS occurred in lung cancer patients with controlled versus uncontrolled glycemia (P < .0001). This improvement spanned sex, age, smoking status, insurance status, stage, race, BMI, histopathology, and therapy. Survival tree analysis revealed that obese and morbidly obese patients with controlled glycemia had higher lung cancer OS than comparison groups. Conclusion: These findings indicate a need for optimal glycemic control to improve lung cancer OS in diverse populations with diabetes.

Top 100 downregulated genes after anti-PD-L1 treatment.

Supplemental Fig. S4. (A) Effect of engineered KRAS expression on response to CDK2 inhibition by seliciclib. KRAS mutation sensitized lung cancer cells towards seliciclib-mediated CDK2 inhibition of growth as compared to control-ED-1 cells. (B) Cells transfected with the plasmid expressing HA-tagged human CP110 were used for detection of CP110 using an anti-CP110 antibody (1:1000) and an anti-HA antibody (1:1000), respectively. The upper band detected by the anti-CP110 antibody specifically recognizes CP110. (C) Respective CP110 and RAS protein expression profile in murine lung cancer cell lines is shown. (D) Effect of KRAS knockdown at 48 hours (left panel) and 72 hours (right panel) on CP110 expression in the 344P cell line. (E) Effect of KRAS knockdown at 72 hours (left panel) and 96 hours (right panel) on CP110 expression in the Hop62 cell line.

Supplementary Table 1 from High DNA Methyltransferase 3B Expression Mediates 5-Aza-Deoxycytidine Hypersensitivity in Testicular Germ Cell Tumors

Supplementary Figure 4 from Hedgehog-Producing Cancer Cells Respond to and Require Autocrine Hedgehog Activity

Supplementary Figure 3 from Hedgehog-Producing Cancer Cells Respond to and Require Autocrine Hedgehog Activity

Supplemental Table 2. Lung cancer patient characteristics in the MedStar-Georgetown University cohort. All lung cancers and those with adenocarcinoma (AD) or squamous cell cancer (SCC) histopathology are shown.

PDF file - 328K, Deficient CD8+ T cell expansion in response to OVA, alpha-CD40 and pI:C immunization in dnRARalphaCD4Cre mice is independent of CD4+ T cells

Genes with altered expression in G0S2 null MEFs as compared to wild-type MEFs

PDF file, 90K, Repression of UBP43 mRNA levels reduces cyclin D1 protein, but not mRNA expression.