Advanced Cancer Therapeutics

Abstract LMO2 is an intrinsically disordered transcription factor activated in T cell leukaemia that is difficult to target. It forms part of a multiprotein complex that has bipartite DNA binding through heterodimeric bHLH and GATA proteins. To determine if degradation of LMO2 in the context of T-ALL has therapeutic potential, a chimaeric intracellular antibody has been developed fusing an anti-LMO2 single domain variable region with one of three E3 ligases to create biodegraders. The intracellular binary interaction of these biodegraders with LMO2 leads to its proteosomal degradation but, in addition, concomitant loss of bHLH proteins that associate with LMO2 in the DNA-binding complex. Chemical compound surrogates of the intracellular antibody paratope (called Abd compounds) have been modified to create proteolysis targeting chimaeras (PROTACs) for orthogonal assays of effects of LMO2 degradation. These form a ternary complex with LMO2 and E3 ligase in leukaemia cells that induces degradation of LMO2, and is also accompanied by loss of associated bHLH proteins. This is accompanied by T-ALL growth inhibition, alterations in proteins involved in cell cycling and instigation of apoptosis. These effects do not occur in the absence of LMO2. Our work demonstrates that degradation of LMO2 affects T-ALL and the lead compounds can eventually be developed into drugs for patient treatment. Our work describes methods for drug discovery starting with antibody fragments.

In the Research Article “mTORC1 Inhibition Is Required for Sensitivity to PI3K p110α Inhibitors in PIK3CA-Mutant Breast Cancer,” the authors have provided additional competing interests upon request by the editors, and the competing interests statements have been updated. The PDF and HTML (full text) have been corrected.

Abstract Polymorphism within the T cell receptor beta variable gene (TRBV) has been implicated in autoimmune disease and immuneCrelated adverse events (IRAEs) during immunotherapy. Previous efforts to evaluate TRBV polymorphism by whole genome sequencing (WGS) have been hampered by the repetitive nature of the TCRB locus. We present a novel longCamplicon TCRB repertoire sequencing approach to evaluate TRBV polymorphism from peripheral blood, which we use to identify TRBV allele haplotypes in 81 Caucasians.

The advent of targeted therapies has established new standards of care for defined molecular subsets of non-small cell lung cancer (NSCLC). Not only has this led to significant changes in the routine clinical management of lung cancer e.g., multiplexed genomic testing, but it has provided important principles and benchmarks for determining "actionability". At present, the clinical paradigms are most evolved for EGFR mutations and ALK rearrangements, where multiple randomized phase III trials have determined optimal treatment strategies in both treatment naïve and resistant settings. However, this may not always be feasible with low prevalence alterations e.g., ROS1 and BRAF mutations. Another emerging observation is that not all targets are equally "actionable", necessitating a rigorous preclinical, clinical and translational framework to prosecute new targets and drug candidates. In this review, we will cover the role of targeted therapies for NSCLC harbouring BRAF, MET, HER2 and RET alterations, all of which have shown promise in non-squamous non-small cell lung cancer (ns-NSCLC). We further review some early epigenetic targets in NSCLC, an area of emerging interest. With increased molecular segmentation of lung cancer, we discuss the upcoming challenges in drug development and implementation of precision oncology approaches, especially in light of the complex and rapidly evolving therapeutic landscape.

The advent of next-generation sequencing (NGS) platforms in the realm of clinical molecular diagnostics provides multigene mutational profiling through massively parallel sequencing. We analyzed 415 breast carcinoma samples from 354 patients using NGS in known hotspots of 46 commonly known cancer-causing genes. A total of 281 somatic nonsynonymous mutations were detected in 62.1% of patients. TP53 was most frequently mutated (38.8%), followed by PIK3CA (31.7%), AKT1 (6%), and ATM (3.9%), with other mutations detected at a lower frequency. When stratified into clinically relevant therapeutic groups (estrogen receptor [ER]/progesterone receptor [PR]+ human epidermal growth factor receptor 2 [HER2]-, ER/PR+HER2+, ER/PR-HER2+, ER/PR/HER2-), each group showed distinct mutational profiles. The ER/PR+HER2- tumors (n = 132) showed the highest frequency of PIK3CA mutations (38%), while the triple-negative tumors (n = 64) had a significantly higher number of TP53 mutations (62%). Of the 61 patients tested for both primary and metastatic tumors, concordant results were seen in 47 (77%) patients, while 13 patients showed additional mutations in the metastasis. Our results indicate that breast cancers may harbor potentially actionable mutations for targeted therapeutics. Therefore, NGS-based mutational profiling can provide useful information that can guide targeted cancer therapy.

Abstract Targeted cancer therapies that block the growth and spread of cancer by interfering with specific onco-proteins are limited by signaling mechanisms that drive intrinsic and acquired resistance. In particular, the MAPK and the PI3K/AKT pathways have been found to both reduce response rates to these agents and to mediate the acquisition of resistance via mutations that activate both pathways. For example, two recently approved BRAFV600E inhibitors, vemurafenib and dabrafenib, cause dramatic clinical responses in ∼50% of BRAFV600E + melanoma patients but universally become ineffective within 5-6 months as a result of genetic alterations that activate the MAPK and PI3K-AKT pathways, including amplifications in BRAFV600E, loss of PTEN, and activating mutations in NRAS and KRAS, MEK, AKT1/3, PIK3CA and PIK3CG. Several of these mutated signaling proteins stimulate glucose metabolism, required for survival and proliferation, in part by increasing the expression and activity of 6-phosphofructo-2-kinase (PFKFB3). For example, PFKFB3 transcription is induced by HIF-1α (which is increased by BRAFV600E, RAS and MEK), loss of PTEN, and PFKFB3 activity is stimulated by AKT via phosphorylation of serine 461. We postulated that PFKFB3 is an essential downstream target of targeted cancer therapies and that the multitude of mutations and amplifications in signaling pathways that cause resistance to these agents activate PFKFB3. In new studies, we demonstrate that BRAFV600E, estradiol and epidermal growth factor each regulate PFKFB3 expression in melanoma cells, breast cancer cells and NSCLC cells, respectively. Moreover, we find that simultaneous inhibition of these oncoproteins or their co-ligands (i.e. the estrogen and EGF receptors) and PFKFB3 using a novel PFKFB3 inhibitor currently in a phase 1 trial, PFK-158, causes a synergistic increase in apoptosis and cytotoxicity in vitro, suggesting that PFK-158 can overcome the intrinsic resistance to these agents. We then decided to determine if these significant synergies would translate in vivo by investigating them in the A375 and A2058 melanoma preclinical models, and in the MCF-7 ER-dependent breast model. For instance, we compared the anti-tumor effects in A375 melanoma xenograft-bearing mice of: (i) the BRAFV600E inhibitor vemurafenib; (ii) PFK-158; or (iii) the combination of vemurafenib and PFK-158. Although we observed significant tumor growth inhibition with both monotherapies (>80%), we only observed tumor regression with the combination therapy (>50%). Results in these different models will be presented. Taken together, these data demonstrate that the PFKFB3 inhibitor PFK-158 may be able to universally overcome resistance to targeted cancer therapies. Furthermore, we predict that phase 1/2 trials of PFK-158 in combination with targeted cancer agents will yield improvements in objective response rates as well as improvements in progression-free survival. Citation Format: Sucheta Telang, Julie O'Neal, Yoannis Imbert-Fernandez, Brian Clem, Nadiia Lypova, Gilles H. Tapolsky, John Trent, Jason Chesney. 6-Phosphofructo-2-Kinase (PFKFB3): At the crossroads of resistance to targeted cancer therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4478. doi:10.1158/1538-7445.AM2015-4478

Abstract Metabolic alterations in cancer are increasingly being recognized as important novel targets. Among different regulators of altered metabolism, 6-PhosphoFructo-2-Kinase/Fructose-2,6-Bisphosphatases 3 (PFKFB3) a glycolytic enzyme required for tumorigenic growth, and produces fructose-2,6-bisphosphate (F2,6BP), is recognized as important regulator of glycolysis. We determined that the active form of PFKFB3ser471 was overexpressed in chemo-resistant ovarian cancer (OVCA) cell lines and resistant patient derived xenograft (PDX) models compared to their isogenic chemo-sensitive cell lines and sensitive PDX models. These results suggest that targeting PFKFB3 will have beneficial effects in patients with OVCA. PFK-158 is a potent and selective inhibitor of PFKFB3 that is currently being investigated in a Phase 1 study in patients with advanced solid malignancies. Here we report for the first time, PFK-158 dose-dependently inhibits cell proliferation and colony formation ability in both carboplatin and paclitaxel (PTX) resistant cell lines. PFK-158 markedly suppresses glycolysis by reducing glucose uptake, ATP production, lactate dehydrogenase activity and lactate secretion. Interestingly, both Chou-Talalay synergy analysis and apoptosis assay by annexin V-PI staining showed PFK-158 strongly synergies with both PTX and carboplatin in PTX and cisplatin resistant cell lines respectively. PFK-158 induced apoptosis through a significant increase in caspase-3/7 activation and PARP cleavage. Moreover, in a highly metastatic PTX-resistant in vivo model, combination of PFK-158 with carboplatin and PTX significantly reduced tumor weight, ascites and metastasis compared to untreated and PFK-158, carboplatin or PTX alone treated mice. Since the majority of OVCA patients recur with chemo-resistant disease, our results suggest that PFK-158 in combination with standard chemotherapy may have clinical utility in late stage ovarian cancer patients. Citation Format: Susmita Mondal, Debarshi Roy, Eleftheria Kalogera, Ashwani Khurana, Gilles H. Tapolsky, Sucheta Telang, Jason Chesney, Viji Shridhar. Inhibition of PFKFB3/glycolysis overcomes chemoresistance in ovarian cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2706. doi:10.1158/1538-7445.AM2015-2706

To improve our understanding of the biological relationships among different types of cancer, we have characterized variation in gene expression patterns in a set of 1,707 samples representing 6 human cancer types (breast, ovarian, brain, colorectal, lung adenocarcinoma and squamous cell lung cancer). In the unified dataset, breast tumors of the Basal-like subtype were found to represent a unique molecular entity as any other cancer type, including the rest of breast tumors, while showing striking similarities with squamous cell lung cancers. Moreover, gene signatures tracking various cancer- and stromal-related biological processes such as proliferation, hypoxia and immune activation were found expressed similarly in different proportions of tumors across the various cancer types. These data suggest that clinical trials focusing on tumors with common profiles and/or biomarker expression rather than their tissue of origin are warranted with a special focus on Basal-like breast cancer and squamous cell lung carcinoma.