Search

Tumor suppressor TP53 is an important gene in human cancer because it is mutated in the majority of tumors, leading to loss-of-function or gain-of-function phenotypes. Mutated TP53 acts like an oncogene, driving cancer progression and causing poor patient outcomes. The role of mutated p53 in cancer has been known for over three decades, yet there is no FDA-approved drug to address the problem. This brief historical perspective highlights some of the insightful advances as well as challenges in therapeutic targeting of p53, especially the mutated forms. The article focuses on a functional p53 pathway restoration approach to drug discovery that years ago was not mainstream, encouraged by anyone, taught in textbooks, or embraced by medicinal chemists. With some knowledge, a clinician scientist's interest, and motivation, the author pursued a unique line of investigation leading to insights for functional bypass of TP53 mutations in human cancer. Like mutated Ras proteins, mutant p53 is fundamentally important as a therapeutic target in cancer and probably deserves a "p53 initiative" like the NCI's "Ras initiative.” There is a link between naivete and enthusiasm for pursuing difficult problems, but important solutions are discovered through hard work and persistence. Hopefully, some benefit comes to patients with cancer from such drug discovery and development efforts.

The discovery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) along with its potent and selective antitumor effects initiated a decades-long search for therapeutic strategies to target the TRAIL pathway. First-generation approaches were focused on the development of TRAIL receptor agonists (TRAs), including recombinant human TRAIL (rhTRAIL) and TRAIL receptor-targeted agonistic antibodies. While such TRAIL pathway-targeted therapies showed promise in preclinical data and clinical trials have been conducted, none have advanced to FDA approval. Subsequent second-generation approaches focused on improving upon the specific limitations of first-generation approaches by ameliorating the pharmacokinetic profiles and agonistic abilities of TRAs as well as through combinatorial approaches to circumvent resistance. In this review, we summarize the successes and shortcomings of first- and second-generation TRAIL pathway-based therapies, concluding with an overview of the discovery and clinical introduction of ONC201, a compound with a unique mechanism of action that represents a new generation of TRAIL pathway-based approaches. We discuss preclinical and clinical findings in different tumor types and provide a unique perspective on translational directions of the field.

PurposeGene fusions involving receptor tyrosine kinases (RTKs) define an important class of genomic alterations with many successful targeted therapies now approved for ALK, ROS1, RET and NTRK gene fusions. Fusions involving the ERBB family of RTKs have been sporadically reported, but their frequency has not yet been comprehensively analyzed and functional characterization is lacking on many types of ERBB fusions.Materials and methodsWe analyzed tumor samples submitted to Caris Life Sciences (n=64,354), as well as the TCGA (n=10,967), MSK IMPACT (n=10,945) and AACR GENIE (n=96,324) databases for evidence of EGFR, ERBB2 and ERBB4 gene fusions. We also expressed several novel fusions in cancer cell lines and analyzed their response to EGFR and HER2 tyrosine kinase inhibitors (TKIs).ResultsIn total, we identified 1,251 ERBB family fusions, representing an incidence of approximately 0.7% across all cancer types. EGFR, ERBB2, and ERBB4 fusions were most frequently found in glioblastoma, breast cancer and ovarian cancer, respectively. We modeled two novel types of EGFR and ERBB2 fusions, one with a tethered kinase domain and the other with a tethered adapter protein. Specifically, we expressed EGFR-ERBB4, EGFR-SHC1, ERBB2-GRB7 and ERBB2-SHC1, in cancer cell lines and demonstrated that they are oncogenic, regulate downstream signaling and are sensitive to small molecule inhibition with EGFR and HER2 TKIs.ConclusionsWe found that ERBB fusions are recurrent mutations that occur across multiple cancer types. We also establish that adapter-tethered and kinase-tethered fusions are oncogenic and can be inhibited with EGFR or HER2 inhibitors. We further propose a nomenclature system to categorize these fusions into several functional classes.

Sup Tables 5-7- Amino Acid Biochrom

Despite significant treatment advances over the past decade, multiple myeloma (MM) remains largely incurable. In this study we found that MM cells were remarkably sensitive to the death-inducing effects of a new class of sangivamycin-like molecules (SLM). A panel of structurally related SLMs selectively induced apoptosis in MM cells but not other tumor or nonmalignant cell lines at submicromolar concentrations. SLM6 was the most active compound in vivo, where it was well tolerated and significantly inhibited growth and induced apoptosis of MM tumors. We determined that the anti-MM activity of SLM6 was mediated by direct inhibition of cyclin-dependent kinase 9 (CDK9), which resulted in transcriptional repression of oncogenes that are known to drive MM progression (MAF, CCND1, MYC, and others). Furthermore, SLM6 showed superior in vivo anti-MM activity more than the CDK inhibitor flavopiridol, which is currently in clinical trials for MM. These findings show that SLM6 is a novel CDK9 inhibitor with promising preclinical activity as an anti-MM agent. Mol Cancer Ther; 11(11); 2321–30. ©2012 AACR.

Supplementary Figure Legends

Tumor microenvironment in GSK-3B-mutated tumors across histologies.

Supplementary Figure from Small-Molecule NSC59984 Induces Mutant p53 Degradation through a ROS-ERK2-MDM2 Axis in Cancer Cells

Figure S1. A. sg12 successfully edited the CDKN1A gene in SW780 with high efficiency; chromatogram shows deletion of an adenine (A). B. Histograms from TIDE analysis of single-cell clones showed that the clones have largely homogeneous populations. Figure S2. Cisplatin sensitivity was assessed in SW780 CRISPR clones 24h after a 3h pulse dose. Increased PARP cleavage (a marker of apoptosis) was observed in three p21-deficient sg12 clones compared to sgGFP. Figure S3: see separate document. Figure S4. TCC cell lines harboring p53 mutations differentially regulate p21 after cisplatin treatment (10 µM 3-hour pulse dose) Table S1: Primary antibodies used Table S2: Secondary antibodies used Table S3: sgRNA and flanking primer sequences

PDF file, 55K.

TMA Clinical Data

PDF file, 30K, Combining DR4 Atrimer� complexes with TRAIL. Sub-G1 content in HCT116 cells treated with 1G2 (50 ng/mL) or TRAIL (25 ng/mL) for 24 hours (n=2).

PDF file, 77K, SLM6 shows combinatorial activity with bortezomib.

RNAseq

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell–mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC.Significance:This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211

PDF file, 84K, Excess soluble DR4, but not DR5, blocks the cytotoxic effect of agonist DR4 Atrimer � complexes. Atrimer� complex 1C9 was pre-incubated with or without soluble DR4-Fc or DR5-Fc for 1 hour before addition to Colo205 cells. Cell viability was measured at 48 hours using the ViaLight Plus kit.

Supplementary Methods

GSK-3B mutated tumors and PD-L1 expression - non-significant histologies

PDF file, 121K, Screening for cytotoxicity of SLM3 in cancer cell lines derived from multiple tumor types.

TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G2, showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL. Mol Cancer Ther; 11(10); 2087–95. ©2012 AACR.

Figure S3: SW780 and RT4 cells are sensitized to cisplatin after the loss of p21. SW780 sg12A clone is more sensitive to cisplatin than sg109A clone, whereas in RT4, the sg12A and sg109a clones have similar sensitivities to cisplatin.

Amino Acid- Mass Spec

Microarray

Supplementary Figure 1. UPR genes in DU145 cells. Supplementary Figure 2. ONC201 does not inhibit translocation of AR-GFP to the nucleus upon DHT treatment and does not induce AR degradation. Supplementary Figure 3. ONC201 partly synergizes with docetaxol. Supplementary Figure 4. ONC201 partly synergizes with crizotinib. Supplementary Figure 5. Biweekly measurements of mouse weights after the indicated treatments in different prostate cancer xenograft models.

TMA IHC Scores

Supplementary Table from Small-Molecule NSC59984 Induces Mutant p53 Degradation through a ROS-ERK2-MDM2 Axis in Cancer Cells

PDF file, 107K, SLM6 is a potent and direct inhibitor of CDK9, CDK1, and CDK2.

PDF file, 80K, Schematic representation of different library designs for primary and affinity matured selections. The native sequence of the tetranectin loop regions is shown at the top. Alterations in the primary and affinity maturation libraries are shown below where an x indicates a position that was randomized in each library.

Supplementary figure 1: Replication of recombinant adenoviruses in MCF7 breast cancer cells. Supplementary figure 2: Telomerase expression in normal human cells and cancer cell lines. Supplementary figure 3: Labeling mixtures of HCT116 colon cancer cells and WBCs. Supplementary figure 4: Imaging the mixture of HCT116 colon cancer cells and WBCs. Supplementary figure 5: Imaging the mixture of MCF7 breast cancer cells and WBCs.

18 supplementary figures and corresponding legends

PDF file, 124K, Comparison of MM oncoprotein and housekeeper protein half-lives.

PDF file, 90K, GSK-3β and CDK1 inhibitors fail to reduce MM cell viability.

Androgen receptor (AR) signaling plays a key role in prostate cancer progression, and androgen deprivation therapy (ADT) is a mainstay clinical treatment regimen for patients with advanced disease. Unfortunately, most prostate cancers eventually become androgen-independent and resistant to ADT with patients progressing to metastatic castration-resistant prostate cancer (mCRPC). Constitutively activated AR variants (AR-V) have emerged as mediators of resistance to AR-targeted therapy and the progression of mCRPC, and they represent an important therapeutic target. Out of at least 15 AR-Vs described thus far, AR-V7 is the most abundant, and its expression correlates with ADT resistance. ONC201/TIC10 is the founding member of the imipridone class of small molecules and has shown anticancer activity in a broad range of tumor types. ONC201 is currently being tested in phase I/II clinical trials for advanced solid tumors, including mCRPC, and hematologic malignancies. There has been promising activity observed in patients in early clinical testing. This study demonstrates preclinical single-agent efficacy of ONC201 using in vitro and in vivo models of prostate cancer. ONC201 has potent antiproliferative and proapoptotic effects in both castration-resistant and -sensitive prostate cancer cells. Furthermore, the data demonstrate that ONC201 downregulates the expression of key drivers of prostate cancer such as AR-V7 and downstream target genes including the clinically used biomarker PSA (KLK3). Finally, the data also provide a preclinical rationale for combination of ONC201 with approved therapeutics for prostate cancer such as enzalutamide, everolimus (mTOR inhibitor), or docetaxel.Implications: The preclinical efficacy of ONC201 as a single agent or in combination, in hormone-sensitive or castration-resistant prostate cancer, suggests the potential for immediate clinical translation. Mol Cancer Res; 16(5); 754–66. ©2018 AACR.

Association between mismatch repair deficiency and differences in PD-L1 expression between GSK-3B-mutated and GSK-3B-wild type tumors

Lists all key resources and reagents

PDF file, 55K, Surface DR4 knockdown. Surface DR4 by flow cytometry in HCT116 cells transfected with a scramble control or DR4 siRNA.

Supplementary Table S1: list of antibodies used in this study for western blots (WB), immunofluorescence (IF) and immunohistochemistry (IHC.)

Supplementary Table S2: differential blood cell count of mice treated with ONC212, 2-DG, or the combination of both agents.

Supplementary Table S3: blood chemistry results of mice treated with ONC212 or the combination of ONC212 and 2-DG

PDF file, 157K, Representative example of screening affinity matured Atrimer� complexes. Single point cytotoxicity assays (A) we performed with 293 PEAK supernatants at 1:2 and 1:10 dilution using the Vialight Plus kit and compared to previously identified agonists. DR4 ELISA (B) was performed concurrently with the same supernatants diluted 1:20 and also compared to previously identified binders.