Sumie Koike

Two novel ALK mutations mediate acquired resistance to the next generation ALK inhibitor alectinib

Purpose: The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged non–small cell lung cancer (NSCLC). Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance. Experimental Design: We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure–activity relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE. Results: We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib. Conclusions: We have identified two novel ALK mutations arising after alectinib exposure that are sensitive to other next-generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs. Clin Cancer Res; 20(22); 5686–96. ©2014 AACR.

Platelets Promote Tumor Growth and Metastasis via Direct Interaction between Aggrus/Podoplanin and CLEC-2

The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus–CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus–CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet–tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.

Cabozantinib Overcomes Crizotinib Resistance in ROS1 Fusion–Positive Cancer

Purpose: ROS1 rearrangement leads to constitutive ROS1 activation with potent transforming activity. In an ongoing phase I trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib shows remarkable initial responses in patients with non–small cell lung cancer (NSCLC) harboring ROS1 fusions; however, cancers eventually develop crizotinib resistance due to acquired mutations such as G2032R in ROS1. Thus, understanding the crizotinib-resistance mechanisms in ROS1-rearranged NSCLC and identification of therapeutic strategies to overcome the resistance are required. Experimental Design: The sensitivity of CD74–ROS1–transformed Ba/F3 cells to multiple ALK inhibitors was examined. Acquired ROS1 inhibitor–resistant mutations in CD74–ROS1 fusion were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3 cells. To overcome the resistance mutation, we performed high-throughput drug screening with small-molecular inhibitors and anticancer drugs used in clinical practice or being currently tested in clinical trials. The effect of the identified drug was assessed in the CD74–ROS1–mutant Ba/F3 cells and crizotinib-resistant patient-derived cancer cells (MGH047) harboring G2032R-mutated CD74–ROS1. Results: We identified multiple novel crizotinib-resistance mutations in the ROS1 kinase domain, including the G2032R mutation. As the result of high-throughput drug screening, we found that the cMET/RET/VEGFR inhibitor cabozantinib (XL184) effectively inhibited the survival of CD74–ROS1 wild-type (WT) and resistant mutants harboring Ba/F3 and MGH047 cells. Furthermore, cabozantinib could overcome all the resistance by all newly identified secondary mutations. Conclusions: We developed a comprehensive model of acquired resistance to ROS1 inhibitors in NSCLC with ROS1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance. Clin Cancer Res; 21(1); 166–74. ©2014 AACR.

Dofequidar fumarate sensitizes cancer stem-like side population cells to chemotherapeutic drugs by inhibiting ABCG2/BCRP-mediated drug export

The ATP‐binding cassette (ABC) transporters (ABC‐T) actively efflux structurally and mechanistically unrelated anticancer drugs from cells. As a consequence, they can confer multidrug resistance (MDR) to cancer cells. ABC‐T are also reported to be phenotypic markers and functional regulators of cancer stem/initiating cells (CSC) and believed to be associated with tumor initiation, progression, and relapse. Dofequidar fumarate, an orally active quinoline compound, has been reported to overcome MDR by inhibiting ABCB1/P‐gp, ABCC1/MDR‐associated protein 1, or both. Phase III clinical trials suggested that dofequidar had efficacy in patients who had not received prior therapy. Here we show that dofequidar inhibits the efflux of chemotherapeutic drugs and increases the sensitivity to anticancer drugs in CSC‐like side population (SP) cells isolated from various cancer cell lines. Dofequidar treatment greatly reduced the cell number in the SP fraction. Estimation of ABC‐T expression revealed that ABCG2/breast cancer resistance protein (BCRP) mRNA level, but not the ABCB1/P‐gp or ABCC1/MDR‐associated protein 1 mRNA level, in all the tested SP cells was higher than that in non‐SP cells. The in vitro vesicle transporter assay clarified that dofequidar had the ability to suppress ABCG2/BCRP function. Dofequidar treatment sensitized SP cells to anticancer agents in vitro. We compared the antitumor efficacy of irinotecan (CPT‐11) alone with that of CPT‐11 plus dofequidar in xenografted SP cells. Although xenografted SP tumors showed resistance to CPT‐11, treatment with CPT‐11 plus dofequidar greatly reduced the SP‐derived tumor growth in vivo. Our results suggest the possibility of selective eradication of CSC by inhibiting ABCG2/BCRP. (Cancer Sci 2009)

Transforming growth factor-β decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells

Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characterized the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-β (TGF-β) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-β repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-β, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-β negatively contributes to maintain the CICs within the cancer.

PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has selective antitumor activity. Although TNF-α-induced intracellular signaling pathways have been well studied, TRAIL signaling is not fully understood. Here, we identified a novel TRAIL receptor-binding protein, protein arginine methyltransferase 5 (PRMT5), as a result of proteomic screening. PRMT5 selectively interacted with death receptor 4 and death receptor 5 but not with TNF receptor 1 or Fas. PRMT5 gene silencing sensitized various cancer cells to TRAIL without affecting TRAIL resistance in nontransformed cells. PRMT5 contributed to TRAIL-induced activation of inhibitor of κB kinase (IKK) and nuclear factor-κB (NF-κB), leading to induction of several NF-κB target genes. Although IKK inhibition increased sensitivity to both TRAIL and TNF-α, PRMT5 knockdown potentiated TRAIL-mediated cytotoxicity alone. PRMT5 had no effect on TNF-α-mediated NF-κB signaling. These results show the selectivity of PRMT5 for TRAIL signaling. The PRMT5 small interfering RNA-mediated susceptibility to TRAIL was rescued by ectopic expression of active IKKβ, confirming the involvement of PRMT5 in TRAIL resistance by activating the NF-κB pathway. Collectively, our findings suggest the therapeutic potential of PRMT5 in TRAIL-based cancer treatments.(Mol Cancer Res 2009;7(4):557–69)

AP-1-Dependent miR-21 expression contributes to chemoresistance in cancer stem cell-like SP cells.

The side population (SP) of cancer cells is a minor population of cells that has been identified in a variety of cancers and harbors many cancer stem cell (CSC)-like properties, such as self-renewal potential, tumorforming capacity, and chemoresistant phenotype. CSCs are regarded as the root of cancer origin and recurrence. Thus, new therapeutic approaches targeting these malignant cells have become the topic of ongoing research. However, the chemoresistant phenotype of CSCs makes it difficult to increase their sensitivity to anticancer drugs and to decrease the rate of cancer recurrence in patients. In this study, we analyzed the chemoresistant phenotype of SP cells derived from various cancer cell lines. Microarray analysis discriminated differential gene expression profiles between SP and non-SP cells. MicroRNA-21 (miR-21) and its upstream regulator activator protein-I (AP-1), composed of c-Jun and c-Fos family transcription factors, were found to be frequently upregulated in SP cells. Downregulation of tumor suppressor programmed cell death 4, one of the miR-21 target gene products, confirmed miR-21 overexpression in SP cells. Treatment of the cells with the AP-1 inhibitor SP600125 attenuated miR-21 levels and increased topotecan sensitivity. Furthermore, specific inhibition of miR-21 by an anti-miR-21 locked nucleic acid increased drug sensitivity and decreased colony forming ability. These findings define the critical role of miR-21 in maintenance of the chemoresistant phenotype of SP cells. Targeting miR-21 may provide a new strategy for cancer therapy by impairing resistance to chemotherapy in CSCs.

P-glycoprotein Mediates Ceritinib Resistance in Anaplastic Lymphoma Kinase-rearranged Non-small Cell Lung Cancer

The anaplastic lymphoma kinase (ALK) fusion oncogene is observed in 3%–5% of non-small cell lung cancer (NSCLC). Crizotinib and ceritinib, a next-generation ALK tyrosine kinase inhibitor (TKI) active against crizotinib-refractory patients, are clinically available for the treatment of ALK-rearranged NSCLC patients, and multiple next-generation ALK-TKIs are currently under clinical evaluation. These ALK-TKIs exhibit robust clinical activity in ALK-rearranged NSCLC patients; however, the emergence of ALK-TKI resistance restricts the therapeutic effect. To date, various secondary mutations or bypass pathway activation-mediated resistance have been identified, but large parts of the resistance mechanism are yet to be identified. Here, we report the discovery of p-glycoprotein (P-gp/ABCB1) overexpression as a ceritinib resistance mechanism in ALK-rearranged NSCLC patients. P-gp exported ceritinib and its overexpression conferred ceritinib and crizotinib resistance, but not to PF-06463922 or alectinib, which are next-generation ALK inhibitors. Knockdown of ABCB1 or P-gp inhibitors sensitizes the patient-derived cancer cells to ceritinib, in vitro and in vivo. P-gp overexpression was identified in three out of 11 cases with in ALK-rearranged crizotinib or ceritinib resistant NSCLC patients. Our study suggests that alectinib, PF-06463922, or P-gp inhibitor with ceritinib could overcome the ceritinib or crizotinib resistance mediated by P-gp overexpression.

Prevention of hematogenous metastasis by neutralizing mice and its chimeric anti-Aggrus/podoplanin antibodies

The platelet aggregation‐inducing factor, Aggrus (also known as podoplanin), is reported to contribute to cancer metastasis by mediating cancer cell–platelet interaction. Aggrus has been shown to be upregulated in many different types of cancers. Thus, not only the functional inhibition of Aggrus, but also its application as a cancer‐specific antigen has therapeutic potential. Among a series of anti‐Aggrus mAb established previously, no mouse anti‐human Aggrus mAb exists that possesses the ability to neutralize platelet aggregation. For precise preclinical examinations of mouse and monkey models, the establishment of Aggrus‐neutralizing mouse mAb and their chimeric Abs is needed. In this study, we established two mouse anti‐human Aggrus mAb, P2‐0 and HAG‐3. A precise analysis of their epitopes revealed that P2‐0 recognized the conformation near the bioactive O‐glycosylation site at the Thr52 residue. In contrast, HAG‐3 recognized the amino‐terminus side at a short distance from the conformation recognized by P2‐0. We observed that only P2‐0 attenuated Aggrus‐induced platelet aggregation and Aggrus binding to its platelet receptor, that is, the C‐type lectin‐like receptor‐2. Consistent with these data, only P2‐0 prevented the experimental metastasis of human Aggrus‐overexpressing CHO cells. Subsequently, we cloned the complementary determining region of P2‐0 and produced the murine/human chimeric P2‐0 antibody. This chimeric antibody maintained its inhibitory activity of Aggrus‐induced platelet aggregation and experimental metastasis. Thus, P2‐0 and its chimeric antibody are expected to aid the development of preclinical and clinical examinations of Aggrus‐targeted cancer therapy. (Cancer Sci 2011; 102: 2051–2057)

Recurrent 8q24 rearrangement in blastic plasmacytoid dendritic cell neoplasm: association with immunoblastoid cytomorphology, MYC expression, and drug response

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare skin-tropic hematological malignancy of uncertain pathogenesis and poor prognosis. We examined 118 BPDCN cases for cytomorphology, MYC locus rearrangement, and MYC expression. Sixty-two (53%) and 41 (35%) cases showed the classic and immunoblastoid cytomorphology, respectively. Forty-one (38%) MYC+BPDCN (positive for rearrangement and expression) and 59 (54%) MYC−BPDCN (both negative) cases were identified. Immunoblastoid cytomorphology was significantly associated with MYC+BPDCN. All examined MYC+BPDCNs were negative for MYB/MYBL1 rearrangement (0/36). Clinically, MYC+BPDCN showed older onset, poorer outcome, and localized skin tumors more commonly than MYC−BPDCN. MYC was demonstrated by expression profiling as one of the clearest discriminators between CAL-1 (MYC+BPDCN) and PMDC05 (MYC−BPDCN) cell lines, and its shRNA knockdown suppressed CAL-1 viability. Inhibitors for bromodomain and extra-terminal protein (BETis), and aurora kinases (AKis) inhibited CAL-1 growth more effectively than PMDC05. We further showed that a BCL2 inhibitor was effective in both CAL-1 and PMDC05, indicating that this inhibitor can be used to treat MYC−BPDCN, to which BETis and AKis are probably less effective. Our data will provide a rationale for the development of new treatment strategies for patients with BPDCN, in accordance with precision medicine.