Early dynamics of circulating tumor DNA predict clinical response to immune checkpoint inhibitors in metastatic renal cell carcinoma.

OBJECTIVES: Detection of genomic alterations in circulating tumor deoxyribonucleic acid of peripheral blood can guide the selection of systemic therapy in cancer patients. The predictive significance of circulating tumor deoxyribonucleic acid in metastatic renal cell carcinoma remains unclear, especially for patients treated with immune checkpoint inhibitors. METHODS: In this study, we collected plasma samples before and 1 month after commencing nivolumab monotherapy or nivolumab plus ipilimumab therapy from 14 metastatic renal cell carcinoma patients. We performed circulating tumor deoxyribonucleic acid genomic profiling in plasma cell-free deoxyribonucleic acid by next-generation sequencing using a commercially available pan-cancer panel (Guardant360 CDx). Additionally, we also performed whole exome sequencing of tumor tissues and compared the concordance of genomic profiles with circulating tumor deoxyribonucleic acid. RESULTS: Nine patients had circulating tumor deoxyribonucleic acid in pretreatment plasma samples with a total of 20 mutations (15 single nucleotide variants, three insertions/deletions, and two copy number amplification). VHL (30.0%) was the most frequently mutated gene, followed by TP53 (20.0%), and 45.0% of circulating tumor deoxyribonucleic acid mutations were concordant with somatic mutations in tumor tissues. Patients with decreasing circulating tumor deoxyribonucleic acid mutant allele frequency had better progression free survival when compared to those with increasing mutant allele frequency (P = 0.0441). CONCLUSIONS: Our findings revealed that early circulating tumor deoxyribonucleic acid dynamics can serve as a predictive biomarker for response to immune checkpoint inhibitors in metastatic renal cell carcinoma patients.

Detection of ERBB2 (HER2) Gene Amplification Events in Cell-Free DNA and Response to Anti-HER2 Agents in a Large Asian Cancer Patient Cohort.

Background: HER2 antagonists have marked activity and are approved for the treatment of HER2 overexpressing breast and gastric cancers. Recent studies have shown that ERBB2 (HER2) gene amplification and overexpression may also be actionable in other tumor types. Inter- and intratumoral heterogeneity in HER2 status, however, poses a significant challenge in identifying patients that may benefit from HER2-targeted therapies. ERBB2 amplification as identified by circulating cell-free DNA (cfDNA), which circumvents tissue heterogeneity issues, is emerging as a robust biomarker predictive of response to anti-HER2 agents. Here, the prevalence and genomic landscape of ERBB2 alterations detectable by next-generation sequencing (NGS) of cfDNA was evaluated in a large cohort of Asian patients with advanced solid tumors. Methods: Results were queried for consecutive patients (n = 469) tested by a comprehensive 70/73-gene cfDNA NGS assay (Guardant360®) between November 2015 and June 2018. Patients with ERBB2 gene alterations including copy number amplifications (CNAs), single nucleotide variants (SNVs), and insertion-deletions (indels) were identified. Results: ERBB2 alterations were detected in 52 patients (11.1%); ERBB2 SNVs, CNAs, and indels were found in 27 (5.8%), 27 (5.8%), and 10 (2.1%) patients, respectively. ERBB2 amplification was most frequently identified in gastric (21.4%; 6/28), colorectal (11.1%; 5/45), lung (3.9%; 9/231), and breast (3.2%; 1/31) cancer patients. ERBB2 amplification was often mutually exclusive with other oncogenic alterations in gastric (83.3%; 5/6) and colorectal (60%; 3/5) cancer patients. ERBB2 copy number gains were also highest in gastric and colorectal cancers (median 4.8 and 6.6, respectively). We further report two cases of advanced gastric cancer patients, one treatment naïve, and the other having failed four lines of therapy, whose ERBB2 CNAs were identified by cfDNA and derived clinical benefit from HER2-based therapies. Conclusion: Our data indicate that ERBB2 amplification is a common event in solid tumors among Asian cancer patients. High ERBB2 incidence and copy number gains were observed in gastric and colorectal cancer patients, often in the absence of other oncogenic mutations, underscoring its likely role as the driver alteration in those settings. Finally, we show the potential of comprehensive cfDNA testing in identifying patients who are most likely to benefit from HER2-targeted therapies.

Clinical Application of Genomic Profiling With Circulating Tumor DNA for Management of Advanced Non-Small-cell Lung Cancer in Asia.

BACKGROUND: Genomic profiling of cell-free circulating tumor DNA (ctDNA) is a potential alternative to repeat invasive biopsy in patients with advanced cancer. We report the first real-world cohort of comprehensive genomic assessments of patients with non-small-cell lung cancer (NSCLC) in a Chinese population. PATIENTS AND METHODS: We performed a retrospective analysis of patients with advanced or metastatic NSCLC whose physician requested ctDNA-based genomic profiling using the Guardant360 platform from January 2016 to June 2017. Guardant360 includes all 4 major types of genomic alterations (point mutations, insertion-deletion alterations, fusions, and amplifications) in 73 genes. RESULTS: Genomic profiling was performed in 76 patients from Hong Kong during the 18-month study period (median age, 59.5 years; 41 men and 35 women). The histologic types included adenocarcinoma (n = 10), NSCLC, not otherwise specified (n = 58), and squamous cell carcinoma (n = 8). In the adenocarcinoma and NSCLC, not otherwise specified, combined group, 62 of the 68 patients (91%) had variants identified (range, 1-12; median, 3), of whom, 26 (42%) had ≥ 1 of the 7 National Comprehensive Cancer Network-recommended lung adenocarcinoma genomic targets. Concurrent detection of driver and resistance mutations were identified in 6 of 13 patients with EGFR driver mutations and in 3 of 5 patients with EML4-ALK fusions. All 8 patients with squamous cell carcinoma had multiple variants identified (range, 1-20; median, 6), including FGFR1 amplification and ERBB2 (HER2) amplification. PIK3CA amplification occurred in combination with either FGFR1 or ERBB2 (HER2) amplification or alone. CONCLUSION: Genomic profiling using ctDNA analysis detected alterations in most patients with advanced-stage NSCLC, with targetable aberrations and resistance mechanisms identified. This approach has demonstrated its feasibility in Asia.

Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells.

Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5-5 µM induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 µM, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10-20 µM), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM.

Hsp90 inhibitors as anti-cancer agents, from basic discoveries to clinical development.

Heat shock protein (Hsp) 90 is an ATP-dependent molecular chaperone which stabilizes various oncogenic kinases, including HER2, EGFR, BCR-ABL, B-Raf and EML4-ALK, which are essential for tumor growth. Several monoclonal antibodies and small molecule kinase inhibitors which target these kinases have been identified as potential new molecular target therapeutics. Previous reports have shown that many oncogenic proteins essential for cancer transformation are chaperoned by the Hsp90 complex, and some of these client proteins have been discovered by using Hsp90 inhibitors, such as geldanamycin (GA) and radicicol (RD).Thus far more than 200 client proteins have been identified. In past derivatives of these natural products have been evaluated in clinical trials, but none of the 1st generation of Hsp90 inhibitors has been approved yet because of their limitations in physico-chemical properties and/or safety profiles. However, recent reports have indicated that more than 10 new agents, 2nd generation of Hsp90 inhibitors with different chemotypes from GA and RD, have entered clinical trials and some of them showed clinical efficacy. In this review article, we describe the discoveries of major Hsp90 client proteins in the cancer field by RD derivatives, the history of KW-2478 discovery and development by Kyowa Hakko Kirin, and gave an update on the current status of new Hsp90 inhibitors in clinical trials.

RSK2(Ser227) at N-terminal kinase domain is a potential therapeutic target for multiple myeloma.

Multiple myeloma is an entity of cytogenetically and genetically heterogenous plasma cell neoplasms. Despite recent improvement in the treatment outcome of multiple myeloma by novel molecular-targeted chemotherapeutics, multiple myeloma remains incurable. The identification of a therapeutic target molecule in which various signaling for cell-survival converge is a core component for the development of new therapeutic strategies against multiple myeloma. RSK2 is an essential mediator of the ERK1/2 signaling pathway for cell survival and proliferation. In this study, we discovered that RSK2(Ser227), which is located at the N-terminal kinase domain and is one site responsible for substrate phosphorylation, is activated through phosphorylation regardless of the type of cytogenetic abnormalities or upstream molecular signaling in all 12 multiple myeloma-derived cell lines examined and 6 of 9 patient-derived CD138-positive primary myeloma cells. The chemical inhibition of RSK2(Ser227) by BI-D1870 or gene knockdown of RSK2 inhibits myeloma cell proliferation through apoptosis induction, and this anti-myeloma effect was accompanied by downregulation of c-MYC, cyclin D, p21(WAF1/CIP1), and MCL1. RSK2(Ser227) inhibition resulting from BI-D1870 treatment restored lenalidomide-induced direct cytotoxicity of myeloma cells from interleukin-6-mediated cell protection, showed no cross-resistance to bortezomib, and exerted additive/synergistic antiproliferative effects in conjunction with the mTOR, histone deacetylase, and BH3-mimicking BCL2/BCLX(L) inhibitors. These results suggest that RSK2(Ser227) is a potential therapeutic target not only for newly diagnosed but also for patients with later phase multiple myeloma who are resistant or refractory to currently available anti-myeloma therapies.

Selective KIT inhibitor KI-328 and HSP90 inhibitor show different potency against the type of KIT mutations recurrently identified in acute myeloid leukemia.

Activating mutations of KIT play an important role in the pathophysiology of several human malignancies, including acute myeloid leukemia. Activated KIT kinase is therefore a promising molecular target for the treatment of many malignancies harboring KIT activation. Here we examined the potency of a novel KIT inhibitor KI-328 against different types of mutant KIT kinases recurrently identified in AML. KI-328 shows selective potency against KIT kinase for the in vitro kinase assay, and inhibits the growth of wild-type (Wt)- and mutant-KIT-expressing cells, while it has little potency against D816V-KIT. Comparable analysis of several potent KIT inhibitors regarding growth inhibitory effects on a variety of mutant-KIT-expressing cells revealed that multi-kinase inhibitors have the same potency against D816V-KIT as other mutant KITs; however, the predominant potency against D816V-KIT was observed in heat shock protein 90 (HSP90) inhibitors. Furthermore, HSP90 inhibitors suppress the growth of D816V-KIT-expressing cells at the concentration at which the growth of other mutant-KIT-expressing cells is not affected. These results collectively indicated that potent KIT inhibitors have different potency against the type of mutant KIT kinases. Therefore, KIT inhibitors are required to validate potency against several types of mutant KIT kinases for the clinical development.

[Steroid sulfatase inhibitor].

More than 60 percents of breast cancers occur in post menopausal women and most of their initial stages are hormone dependent. For the treatment of estrogen dependent breast tumors, mainly two treatment tools are available, one is selective estrogen receptor modulator (SERM), and the other is aromatase inhibitor (AI). Although these drugs are clinically valuable, the existence of resistant tumors againt these two treatments is one of the most serious matters. The latest studies clarify that to inhibit the local formation of estrogen is more important than to decrease the estrogen dose in plasma. Steroid sulfatase (STS) is mainly expressed in local breast carcinoma tissues and is one of the most promising targets to inhibit the local formation of estrogens.

New molecular and biological mechanism of antitumor activities of KW-2478, a novel nonansamycin heat shock protein 90 inhibitor, in multiple myeloma cells.

PURPOSE: The heat shock protein 90 (Hsp90) plays an important role in chaperoning oncogenic client proteins in multiple myeloma (MM) cells, and several Hsp90 inhibitors have shown antitumor activities both in vitro and in vivo. However the precise mechanism of action of Hsp90 inhibitor in MM has not been fully elucidated. EXPERIMENTAL DESIGN: We evaluated the antitumor activities of KW-2478, a nonansamycin Hsp90 inhibitor, in MM cells with various chromosomal translocations of immunoglobulin heavy chain (IgH) loci both in vitro and in vivo. RESULTS: Our studies revealed that exposure of KW-2478 to MM cells resulted in growth inhibition and apoptosis, which were associated with degradation of well-known client proteins as well as a decrease in IgH translocation products (FGFR3, c-Maf, and cyclin D1), and FGFR3 was shown to be a new client protein of Hsp90 chaperon complex. In addition, KW-2478 depleted the Hsp90 client Cdk9, a transcriptional kinase, and the phosphorylated 4E-BP1, a translational inhibitor. Both inhibitory effects of KW-2478 on such transcriptional and translational pathways were shown to reduce c-Maf and cyclin D1 expression. In NCI-H929 s.c. inoculated model, KW-2478 showed a significant suppression of tumor growth and induced the degradation of client proteins in tumors. Furthermore, in a novel orthotopic MM model of i.v. inoculated OPM-2/green fluorescent protein, KW-2478 showed a significant reduction of both serum M protein and MM tumor burden in the bone marrow. CONCLUSIONS: These results suggest that targeting such diverse pathways by KW-2478 could be a promising strategy for the treatment of MM with various cytogenetic abnormalities.

KW-2449, a novel multikinase inhibitor, suppresses the growth of leukemia cells with FLT3 mutations or T315I-mutated BCR/ABL translocation.

KW-2449, a multikinase inhibitor of FLT3, ABL, ABL-T315I, and Aurora kinase, is under investigation to treat leukemia patients. In this study, we examined its possible modes of action for antileukemic effects on FLT3-activated, FLT3 wild-type, or imatinib-resistant leukemia cells. KW-2449 showed the potent growth inhibitory effects on leukemia cells with FLT3 mutations by inhibition of the FLT3 kinase, resulting in the down-regulation of phosphorylated-FLT3/STAT5, G(1) arrest, and apoptosis. Oral administration of KW-2449 showed dose-dependent and significant tumor growth inhibition in FLT3-mutated xenograft model with minimum bone marrow suppression. In FLT3 wild-type human leukemia, it induced the reduction of phosphorylated histone H3, G(2)/M arrest, and apoptosis. In imatinib-resistant leukemia, KW-2449 contributed to release of the resistance by the simultaneous down-regulation of BCR/ABL and Aurora kinases. Furthermore, the antiproliferative activity of KW-2449 was confirmed in primary samples from AML and imatinib-resistant patients. The inhibitory activity of KW-2449 is not affected by the presence of human plasma protein, such as alpha1-acid glycoprotein. These results indicate KW-2449 has potent growth inhibitory activity against various types of leukemia by several mechanisms of action. Our studies indicate KW-2449 has significant activity and warrants clinical study in leukemia patients with FLT3 mutations as well as imatinib-resistant mutations.

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients.

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17beta-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor beta, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.

A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response.

Internal tandem duplication mutations of FLT3 (FLT3/ITD mutations) are common in acute myeloid leukemia (AML) and confer a poor prognosis. This would suggest that FLT3 is an ideal therapeutic target, but FLT3 targeted therapy has produced only modest benefits in clinical trials. Due to technical obstacles, the assessment of target inhibition in patients treated with FLT3 inhibitors has been limited and generally only qualitative. KW-2449 is a novel multitargeted kinase inhibitor that induces cytotoxicity in Molm14 cells (which harbor an FLT3/ITD mutation). The cytotoxic effect occurs primarily at concentrations sufficient to inhibit FLT3 autophosphorylation to less than 20% of its baseline. We report here correlative data from a phase 1 trial of KW-2449, a trial in which typical transient reductions in the peripheral blast counts were observed. Using quantitative measurement of FLT3 inhibition over time in these patients, we confirmed that FLT3 was inhibited, but only transiently to less than 20% of baseline. Our results suggest that the failure to fully inhibit FLT3 in sustained fashion may be an underlying reason for the minimal success of FLT3 inhibitors to date, and stress the importance of confirming in vivo target inhibition when taking a targeted agent into the clinical setting.

Novel and orally active 5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives as selective FLT3 inhibitors.

5-(1,3,4-Oxadiazol-2-yl)pyrimidine derivative 1 was identified as a new class of FLT3 inhibitor from our compound library. With the aim of enhancement of antitumor activity of 2 prepared by minor modification of 1, structure optimization of side chains at the 2-, 4-, and 5-positions of the pyrimidine ring of 2 was performed to improve the metabolic stability. Introduction of polar substituents on the 1,3,4-oxadiazolyl group contributed to a significant increase in the metabolic stability. As a result, a series of compounds showed increased efficacy against MOLM-13 xenograft model in mice by oral administration.

A novel FLT3 inhibitor FI-700 selectively suppresses the growth of leukemia cells with FLT3 mutations.

PURPOSE: The aim of this study was to evaluate the antileukemia activity of a novel FLT3 kinase inhibitor, FI-700. EXPERIMENTAL DESIGN: The antileukemia activity of FI-700 was evaluated in human leukemia cell lines, mutant or wild-type (Wt)-FLT3-expressing mouse myeloid precursor cell line, 32D and primary acute myeloid leukemia cells, and in xenograft or syngeneic mouse leukemia models. RESULTS: FI-700 showed a potent IC(50) value against FLT3 kinase at 20 nmol/L in an in vitro kinase assay. FI-700 showed selective growth inhibition against mutant FLT3-expressing leukemia cell lines and primary acute myeloid leukemia cells, whereas it did not affect the FLT3 ligand (FL)-driven growth of Wt-FLT3-expressing cells. These antileukemia activities were induced by the significant dephosphorylations of mutant FLT3 and STAT5, which resulted in G(1) arrest of the cell cycle. Oral administration of FI-700 induced the regression of tumors in a s.c. tumor xenograft model and increased the survival of mice in an i.v. transplanted model. Furthermore, FI-700 treatment eradicated FLT3/ITD-expressing leukemia cells, both in the peripheral blood and in the bone marrow. In this experiment, the depletion of FLT3/ITD-expressing cells by FI-700 was more significant than that of Ara-C, whereas bone marrow suppression by FI-700 was lower than that by Ara-C. CONCLUSIONS: FI-700 is a novel and potent FLT3 inhibitor with promising antileukemia activity.

A nonfucosylated anti-HER2 antibody augments antibody-dependent cellular cytotoxicity in breast cancer patients.

PURPOSE: Removal of fucose residues from the oligosaccharides of human antibody is a powerful approach to enhance antibody-dependent cellular cytotoxicity (ADCC), a potential important antitumor mechanism of therapeutic antibodies. To provide clinically relevant evidence of this mechanism, we investigated ADCC of a fucose-negative version of trastuzumab [anti-human epidermal growth factor receptor 2 (HER2) humanized antibody] using peripheral blood mononuclear cells (PBMC) from breast cancer patients as effector cells. EXPERIMENTAL DESIGN: Thirty volunteers, including 20 breast cancer patients and 10 normal healthy control donors, were recruited randomly, and aliquots of peripheral blood were collected. ADCC of commercial trastuzumab (fucosylated) and its fucose-negative version were measured using PBMCs drawn from the volunteers as effector cells and two breast cancer cell lines with different HER2 expression levels as target cells. Relationships between cytotoxicity and characteristics of the patients, such as content of natural killer cells in PBMCs, type of therapy, FCGR3A genotypes, etc. were also analyzed. RESULTS: ADCC was significantly enhanced with the fucose-negative antibody compared with the fucose-positive antibody using PBMCs from either normal donors or breast cancer patients. Enhancement of ADCC was observed irrespective of the various clinical backgrounds of the patients, even in the chemotherapy cohort that presented with a reduced number of natural killer cells and weaker ADCC. CONCLUSIONS: This preliminary study suggests that the use of fucose-negative antibodies may improve the therapeutic effects of anti-HER2 therapy for patients independent of clinical backgrounds.

A novel steroidal selective steroid sulfatase inhibitor KW-2581 inhibits sulfated-estrogen dependent growth of breast cancer cells in vitro and in animal models.

We screened a series of 17beta-(N-alkylcarbamoyl)-estra-1,3,5(10)trine-3-O-sulfamate derivatives, and describe here a potent and selective steroid sulfatase (STS) inhibitor with antitumor effects in breast cancer models in vitro and in vivo. In biochemical assays using crude enzymes isolated from recombinant Chinese hamster ovary cells expressing human arylsulfatses (ARSs), one of the best compounds, KW-2581, inhibited STS activity with an IC(50) of 4.0 nM, while > 1000-fold higher concentrations were required to inhibit the other ARSs. The failure to stimulate the growth of MCF-7 human breast cancer cells as well as in uteri in ovariectomized rats indicated the lack of estrogenicity of this compound. In MCF-7 cells transfected with the STS gene, termed MCS-2 cells, KW-2581 inhibited the growth of cells stimulated by estrone sulfate (E1S) but also 5-androstene-3beta, 17beta-diol 3-sulfate (ADIOLS) and dehydroepiandrostenedione 3-sulfate. We found that oral administration of KW-2581 inhibited both E1S- and ADIOLS-stimulated growth of MCS-2 cells in a mouse hollow fiber model. In a nitrosomethylurea-induced rat mammary tumor model, KW-2581 induced regression of E1S-stimulated tumor growth as effectively as tamoxifen or another STS inhibitor, 667 Coumate. Dose-response studies in the same rat model demonstrated that more than 90% inhibition of STS activity in tumors was necessary to induce tumor shrinkage. STS activity in tumors has well correlated with that in leukocytes, suggesting that STS activity in leukocytes could be used as an easily detectable pharmacodynamic marker. These findings demonstrate that KW-2581 is a candidate for development as a therapeutic agent for the treatment of hormone receptors-positive breast cancer.

[Current screening for molecular target therapy of cancer].

Recent progress in molecular biology and cancer biology has revealed that many molecules, which are heavily involved in the un-limited growth, anti-apoptotic effects and invasion of tumors, would be targets of cancer therapy. Part of the drugs which inhibit these molecules have shown clinical response as well as clinical benefits. In this review article, the summary of mechanism based drug screening for cancer as well as recent status of new molecular target drugs for cancer, are described.