FL118, acting as a 'molecular glue degrader', binds to dephosphorylates and degrades the oncoprotein DDX5 (p68) to control c-Myc, survivin and mutant Kras against colorectal and pancreatic cancer with high efficacy.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), a difficult-to-treat cancer, is expected to become the second-largest cause of cancer-related deaths by 2030, while colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer deaths. Currently, there is no effective treatment for PDAC patients. The development of novel agents to effectively treat these cancers remains an unmet clinical need. FL118, a novel anticancer small molecule, exhibits high efficacy against cancers; however, the direct biochemical target of FL118 is unknown. METHODS: FL118 affinity purification, mass spectrometry, Nanosep centrifugal device and isothermal titration calorimetry were used for identifying and confirming FL118 binding to DDX5/p68 and its binding affinity. Immunoprecipitation (IP), western blots, real-time reverse transcription PCR, gene silencing, overexpression (OE) and knockout (KO) were used for analysing gene/protein function and expression. Chromatin IP was used for analysing protein-DNA interactions. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromid assay and human PDAC/CRC cell/tumour models were used for determining PDAC/CRC cell/tumour in vitro and in vivo growth. RESULTS: We discovered that FL118 strongly binds to dephosphorylates and degrades the DDX5 oncoprotein via the proteasome degradation pathway without decreasing DDX5 mRNA. Silencing and OE of DDX5 indicated that DDX5 is a master regulator for controlling the expression of multiple oncogenic proteins, including survivin, Mcl-1, XIAP, cIAP2, c-Myc and mutant Kras. Genetic manipulation of DDX5 in PDAC cells affects tumour growth. PDAC cells with DDX5 KO are resistant to FL118 treatment. Our human tumour animal model studies further indicated that FL118 exhibits high efficacy to eliminate human PDAC and CRC tumours that have a high expression of DDX5, while FL118 exhibits less effectiveness in PDAC and CRC tumours with low DDX5 expression. CONCLUSION: DDX5 is a bona fide FL118 direct target and can act as a biomarker for predicting PDAC and CRC tumour sensitivity to FL118. This would greatly impact FL118 precision medicine for patients with advanced PDAC or advanced CRC in the clinic. FL118 may act as a 'molecular glue degrader' to directly glue DDX5 and ubiquitination regulators together to degrade DDX5.

Kras mutation subtypes distinctly affect colorectal cancer cell sensitivity to FL118, a novel inhibitor of survivin, Mcl-1, XIAP, cIAP2 and MdmX.

Mutation-activated Kras in cancer cells is a well-known challenging treatment-resistant factor that plays a critical role in treatment resistance. Human colorectal cancer (CRC) has four major Kras mutations; KrasG12D (34.2%), KrasG12V (21%), KrasG13D (20%) and KrasG12C (8.4%). Here, we report that while FL118 (a novel inhibitor of survivin, Mcl-1, XIAP, cIAP2 and MdmX) exhibits high efficacy to kill CRC cells and eliminate CRC tumors, CRC cells/tumors with different Kras mutation subtypes in the defined p53/APC genetic statuses exhibit different sensitivity to FL118 treatment. Using CRC cell lines, SW620 (KrasG12V, mutant p53, mutant APC), DLD-1 (KrasG13D, wild type p53, mutant APC) and SNU-C2B (KrasG12D, mutant p53, wild type APC), we demonstrated that silencing of KrasG12V and KrasG12D using Kras-specific shRNA significantly increased CRC cell IC50, while silencing of KrasG13D decreased the CRC cell IC50. This finding suggests that both KrasG12V and KrasG12D are required for showing higher FL118 efficacy, while the presence of KrasG13D could somehow decrease FL118 efficacy under the defined p53/APC genetic status. Consistent with this notion, silencing of KrasG12V in SW620 cells decreased FL118-induced apoptosis, while silencing of KrasG13D in DLD-1 cells increased the FL118-induced apoptosis. Furthermore, forced expression of KrasG12V in SW620 cells increased FL118-induced apoptosis, while forced expression of KrasG13D in DLD-1 cells decreased FL118-induced apoptosis. Additionally, FL118 induced differential reactive oxygen species (ROS) production in SW620, DLD-1 and SNU-C2B cells. Our in vivo studies in animal models further confirmed that SW620 tumors are the most sensitive tumor to FL118 treatment; SNU-C2B tumors are the second most sensitive tumor to FL118 treatment; and the DLD-1 tumors are the least sensitive tumor. These findings would be useful for predicting FL118 sensitivity to patients' CRC tumors with the defined Kras mutation subtypes under the defined p53/APC genetic status.

Mutant Kras as a Biomarker Plays a Favorable Role in FL118-Induced Apoptosis, Reactive Oxygen Species (ROS) Production and Modulation of Survivin, Mcl-1 and XIAP in Human Bladder Cancer.

Tumor heterogeneity in key gene mutations in bladder cancer (BC) is a major hurdle for the development of effective treatments. Using molecular, cellular, proteomics and animal models, we demonstrated that FL118, an innovative small molecule, is highly effective at killing T24 and UMUC3 high-grade BC cells, which have Hras and Kras mutations, respectively. In contrast, HT1376 BC cells with wild-type Ras are insensitive to FL118. This concept was further demonstrated in additional BC and colorectal cancer cells with mutant Kras versus those with wild-type Kras. FL118 strongly induced PARP cleavage (apoptosis hallmark) and inhibited survivin, XIAP and/or Mcl-1 in both T24 and UMUC3 cells, but not in the HT1376 cells. Silencing mutant Kras reduced both FL118-induced PARP cleavage and downregulation of survivin, XIAP and Mcl-1 in UMUC3 cells, suggesting mutant Kras is required for FL118 to exhibit higher anticancer efficacy. FL118 increased reactive oxygen species (ROS) production in T24 and UMUC3 cells, but not in HT1376 cells. Silencing mutant Kras in UMUC3 cells reduced FL118-mediated ROS generation. Proteomics analysis revealed that a profound and opposing Kras-relevant signaling protein is changed in UMUC3 cells and not in HT1376 cells. Consistently, in vivo studies indicated that UMUC3 tumors are highly sensitive to FL118 treatment, while HT1376 tumors are highly resistant to this agent. Silencing mutant Kras in UMUC3 cell-derived tumors decreases UMUC3 tumor sensitivity to FL118 treatment. Together, our studies revealed that mutant Kras is a favorable biomarker for FL118 targeted treatment.

An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer.

BACKGROUND: Pancreatic cancer is a deadly disease with a very low 5-year patient survival rate of 6-8%. The major challenges of eliminating pancreatic cancer are treatment resistance and stromal barriers to optimal drug access within the tumor. Therefore, effective molecular targeting drugs with high intra-tumor access and retention are urgently needed for managing this devastating disease in the clinic. METHODS: This study has used the following in vitro and in vivo techniques for the investigation of exceptional anticancer drug FL118's efficacy in treatment of resistant pancreatic cancer: cell culture; immunoblotting analysis to test protein expression; DNA sub-G1 flow cytometry analyses to test cell death; MTT assay to test cell viability; pancreatic cancer stem cell assays (fluorescence microscopy tracing; matrigel assay; CD44-positive cell colony formation assay); human luciferase-labeled pancreatic tumor orthotopic animal model in vivo imaging; pancreatic cancer patient-derived xenograft (PDX) animal models; and toxicology studies with immune-competent BALB/cj mice and beagle dogs. RESULTS: Our studies found that FL118 alone preferentially killed cisplatin-resistant cancer cells, while a combination of FL118 with cisplatin synergistically killed resistant pancreatic cancer cells and reduced spheroid formation of treatment-resistant pancreatic cancer stem-like cells. Furthermore, using in vivo-imaging, we found that FL118 in combination with cisplatin strongly inhibited both drug-resistant pancreatic xenograft tumor growth and metastasis. In PDX model, we demonstrated that FL118 alone effectively eliminated PDX tumors, while FL118 in combination with gemcitabine eliminated PDX tumors that showed relative resistance (less sensitivity) to treatment with FL118. These FL118 efficacy results are consistent with our molecular-targeting data showing that FL118 inhibited the expression of multiple antiapoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) and ERCC6, a critical regulator of DNA repair, in treatment-resistant pancreatic stem-like cancer cells. Furthermore, FL118 toxicity studies in BALB/cj mice and beagle dogs indicated that FL118 exhibits favorable hematopoietic and biochemical toxicities. CONCLUSION: Together, our studies suggest that FL118 is a promising anticancer drug for further clinical development to effectively treat drug-resistant pancreatic cancer alone or in combination with other pancreatic cancer chemotherapeutic drugs.

Topoisomerase I (Top1): a major target of FL118 for its antitumor efficacy or mainly involved in its side effects of hematopoietic toxicity?

FL118 is a novel camptothecin (CPT) analogue that possesses exceptional antitumor efficacy in human tumor animal models. To date, two CPT analogues, irinotecan and topotecan, have been approved by the FDA for cancer treatment. FL118 exhibits superior antitumor activity over irinotecan and topotecan, and effectively overcomes the irinotecan- or topotecan-resistant human tumors in animal models. Accordingly, FL118 selectively inhibits the expression of multiple cancer-associated proteins (survivin, Mcl-1, XIAP, cIAP2, MdmX). However, FL118 has hematopoietic toxicity similar to irinotecan and topotecan, suggesting that FL118's hematopoietic toxicity may share a mechanism similar to irinotecan and topotecan. It is known that CPTs including irinotecan, SN-38 (active metabolite of irinotecan) and topotecan are topoisomerase I (Top1) inhibitors. However, the evidence from our studies failed to reveal that FL118 is a better Top1 inhibitor than SN-38. It was documented that Top1 expression level is positively associated with CPTs' sensitivity. Low Top1 expression links to CPTs' resistance. In contrast to these findings, we found that human colorectal tumor sensitivity to FL118 is irrelevant to the expression level of Top1 protein. FL118 can show high antitumor efficacy in Top1-negative tumors, while Top1 highly positive tumors can exhibit FL118 resistance. This suggests that the presence of Top1 target is not critical for FL118 antitumor activity. In other words, targeting Top1 by FL118 may not play a major role for its antitumor efficacy. However, studies indicate that FL118 can bind to, and inhibit Top1 activity. This raises the possibility that inhibition of Top1 by FL118 may predominantly be involved in hematopoietic toxicity, but not in FL118 antitumor activity. In this article, we will summarize existing observations and provide our up-to-date research results to support our opinion on this important topic.

A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells.

Melanoma has the highest mortality rate of all skin cancers and a major cause of treatment failure is drug resistance. Tumors heterogeneity requires novel therapeutic strategies and new drugs targeting multiple pathways. One of the new approaches is targeting the scaffolding function of tumor related proteins such as focal adhesion kinase (FAK). FAK is overexpressed in most solid tumors and is involved in multiple protein-protein interactions critical for tumor cell survival, tumor neovascularization, progression and metastasis. In this study, we investigated the anticancer activity of the FAK scaffold inhibitor C4, targeted to the FAK-VEGFR-3 complex, against melanomas. We compared C4 inhibitory effects in BRAF mutant vs BRAF wild type melanomas. C4 effectively caused melanoma tumor regression in vivo, when administered alone and sensitized tumors to chemotherapy. The most dramatic effect of C4 was related to reduction of vasculature of both BRAF wild type and V600E mutant xenograft tumors. The in vivo effects of C4 were assessed in xenograft models using non-invasive multimodality imaging in conjunction with histologic and molecular biology methods. C4 inhibited cell viability, adhesion and motility of melanoma and endothelial cells, specifically blocked phosphorylation of VEGFR-3 and FAK and disrupted their complexes. Specificity of in vivo effects for C4 were confirmed by a decrease in tumor FAK and VEGFR-3 phosphorylation, reduction of vasculogenesis and reduced blood flow. Our collective observations provide evidence that a small molecule inhibitor targeted to the FAK protein-protein interaction site successfully inhibits melanoma growth through dual targeting of tumor and endothelial cells and is effective against both BRAF wild type and mutant melanomas.

Targeting the C-terminal focal adhesion kinase scaffold in pancreatic cancer.

Preliminary studies in our laboratory have demonstrated the importance of both the NH2 and COOH terminus scaffolding functions of focal adhesion kinase (FAK). Here, we describe a new small molecule inhibitor, C10, that targets the FAK C-terminus scaffold. C10 showed marked selectivity for cells overexpressing VEGFR3 when tested in isogenic cell lines, MCF7 and MCF7-VEGFR3. C10 preferentially inhibited pancreatic tumor growth in vivo in cells with high FAK-Y925 and VEGFR3 expression. Treatment with C10 led to a significant inhibition in endothelial cell proliferation and tumor endothelial and lymphatic vessel density and decrease in interstitial fluid pressure. These results highlight the underlying importance of targeting the FAK scaffold to treat human cancers.

Ovarian cancer spheroid cells with stem cell-like properties contribute to tumor generation, metastasis and chemotherapy resistance through hypoxia-resistant metabolism.

Cells with sphere forming capacity, spheroid cells, are present in the malignant ascites of patients with epithelial ovarian cancer (EOC) and represent a significant impediment to efficacious treatment due to their putative role in progression, metastasis and chemotherapy resistance. The exact mechanisms that underlie EOC metastasis and drug resistance are not clear. Understanding the biology of sphere forming cells may contribute to the identification of novel therapeutic opportunities for metastatic EOC. Here we generated spheroid cells from human ovarian cancer cell lines and primary ovarian cancer. Xenoengraftment of as few as 2000 dissociated spheroid cells into immune-deficient mice allowed full recapitulation of the original tumor, whereas >10(5) parent tumor cells remained non-tumorigenic. The spheroid cells were found to be enriched for cells with cancer stem cell-like characteristics such as upregulation of stem cell genes, self-renewal, high proliferative and differentiation potential, and high aldehyde dehydrogenase (ALDH) activity. Furthermore, spheroid cells were more aggressive in growth, migration, invasion, scratch recovery, clonogenic survival, anchorage-independent growth, and more resistant to chemotherapy in vitro. (13)C-glucose metabolic studies revealed that spheroid cells route glucose predominantly to anaerobic glycolysis and pentose cycle to the detriment of re-routing glucose for anabolic purposes. These metabolic properties of sphere forming cells appear to confer increased resistance to apoptosis and contribute to more aggressive tumor growth. Collectively, we demonstrated that spheroid cells with cancer stem cell-like characteristics contributed to tumor generation, progression and chemotherapy resistance. This study provides insight into the relationship between tumor dissemination and metabolic attributes of human cancer stem cells and has clinical implications for cancer therapy.

The FAK scaffold inhibitor C4 disrupts FAK-VEGFR-3 signaling and inhibits pancreatic cancer growth.

Even with successful surgical resection and perioperative chemotherapy and radiation, pancreatic ductal adenocarcinoma (PDA) has a high incidence of recurrence. Tumor cell survival depends on activation of signaling pathways that suppress the apoptotic stimuli of invasion and metastasis. Focal adhesion kinase (FAK) is a critical signaling molecule that has been implicated in tumor cell survival, invasion and metastasis. We have previously shown that FAK and vascular endothelial growth factor receptor 3 (VEGFR-3) are overexpressed in cancer cells and physically interact to confer a significant survival advantage. We subsequently identified a novel small molecule inhibitor C4 that targeted the VEGFR-3-FAK site of interaction. In this study, we have shown that C4 disrupted the FAK-VEGFR-3 complexes in PDA cells. C4 treatment caused dose-dependent dephosphorylation and inactivation of the VEGFR-3 and FAK, reduction in cell viability and proliferation, cell cycle arrest and apoptosis in PDA cells. C4 increased the sensitivity of tumor cells to gemcitabine chemotherapy in vitro that lead to apoptosis at nanomolar concentrations of both drugs. C4 reduced tumor growth in vivo in subcutaneous and orthotopic murine models of PDA. The drug alone at low dose, decreased tumor growth; however, concomitant administration with low dose of gemcitabine had significant synergistic effect and led to 70% tumor reduction. Combination of C4 with gemcitabine had a prolonged cytostatic effect on tumor growth after treatment withdrawal. Finally, we report an anecdotal case of stage IV pancreatic cancer treated with gemcitabine in combination with C4 that showed a significant clinical response in primary tumor and complete clinical response in liver metastasis over an eight month period. Taken together, these results demonstrate that targeting the scaffolding function of FAK with a small-molecule FAK-VEGFR-3 inhibitor can be an effective therapeutic strategy against PDA.

Effects of 1-methyltryptophan stereoisomers on IDO2 enzyme activity and IDO2-mediated arrest of human T cell proliferation.

IDO2 is a newly discovered enzyme with 43 % similarity to classical IDO (IDO1) protein and shares the same critical catalytic residues. IDO1 catalyzes the initial and rate-limiting step in the degradation of tryptophan and is a key enzyme in mediating tumor immune tolerance via arrest of T cell proliferation. The role of IDO2 in human T cell immunity remains controversial. Here, we demonstrate that similar to IDO1, IDO2 also degrades tryptophan into kynurenine and is inhibited more efficiently by Levo-1-methyl tryptophan (L-1MT), an IDO1 competitive inhibitor, than by dextro-methyl tryptophan (D-1MT). Although IDO2 enzyme activity is weaker than IDO1, it is less sensitive to 1-MT inhibition than IDO1. Moreover, our results indicate that human CD4(+) and CD8(+) T cell proliferation was inhibited by IDO2, but both L-1MT and D-1MT could not reverse IDO2-mediated arrest of cell proliferation, even at high concentrations. These data indicate that IDO2 is an inhibitory mechanism in human T cell proliferation and support efforts to develop more effective IDO1 and IDO2 inhibitors in order to overcome IDO-mediated immune tolerance.

Expression and serum immunoreactivity of developmentally restricted differentiation antigens in epithelial ovarian cancer.

Cancer-embryo antigens or developmentally restricted differentiation antigens (DRDAGs), such as PLAC1 (CT92) and developmental pluripotency associated-2 (DPPA2/CT100), are expressed in pluripotent embryonic cells. They are also recognized as cancer-testis antigens (CT) which are proteins normally expressed only in the human germ line but that are also present in a significant subset of malignant tumors. These antigens may prove to be markers of 'repopulating' cells with stem cell-like characteristics and could be critical targets for immunotherapy in epithelial ovarian cancer (EOC). Our objective was to define the frequency of expression and immunogenicity of PLAC1 and DPPA2 in EOC and correlate expression with clinical outcome. One-step reverse transcriptase PCR was performed on 101 EOC samples and a panel of normal tissues. Expression of PLAC1 and DPPA2 in the EOC specimens was 21/101 (21%) and 31/101 (31%) respectively. In normal tissues, PLAC1 expression was restricted to the placenta while DPPA2 expression was restricted to the placenta and testis. Immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) were also performed on a subset of specimens. Humoral immunity was demonstrable in 2/12 serum samples from patients whose tumors expressed DPPA2. There was no demonstrable antibody response to PLAC1 in patients with PLAC1 positive tumors. The presence of PLAC1 and DPPA2 did not have a statistically significant effect on recurrence-free and overall survival. The tissue-restricted expression of PLAC1 and DPPA2, their expression in a significant proportion of EOC patients, and their potential to represent markers of stem cells make DRDAGs attractive targets for antigen-specific immunotherapy in EOC.

Efficacy of levo-1-methyl tryptophan and dextro-1-methyl tryptophan in reversing indoleamine-2,3-dioxygenase-mediated arrest of T-cell proliferation in human epithelial ovarian cancer.

It has been reported that levo-1-methyl tryptophan (L-1MT) can block indoleamine-2,3-dioxygenase (IDO) expressed by human dendritic cells (DC), whereas dextro-1-methyl tryptophan (D-1MT) is inefficient. However, whether L-1MT or D-1MT can efficiently reverse IDO-induced arrest of human T-cell proliferation has not been clarified. Here, we show a marked immunosuppressive effect of IDO derived from INDO-transfected 293 cell, IDO+ ovarian cancer cells, and monocyte-derived DCs on CD4+ Th1 cells, CD8+ T cells, and natural killer cells derived from peripheral blood, ascites, and tumors of ovarian cancer patients. We found that, whereas L-1MT and D/L-1MT can restore proliferation of tumor-derived and peripheral blood T-cell subsets, D-1MT does not effectively restore IDO-induced arrest of T-cell proliferation. Although D-1MT inhibited kynurenine production at high concentrations, L-1MT was more effective in abrogating kynurenine generation and tryptophan depletion, whereas tryptophan was completely depleted by IDO even in the presence of high amounts of D-1MT. Together, the results indicate that, whereas the generation of tryptophan metabolites (kynurenines) by IDO is important in mediating suppression of T-cell proliferation, the degree to which tryptophan depletion is restored by 1MT is also critical in overcoming IDO-induced arrest of T-cell proliferation.