Expanding screening programmes, better cancer diagnostics, breakthrough therapeutics, and biomarker-guided precision oncology have all improved the overall outlook for cancer. However, these advances have not been achieved for all cancers. Advanced hepatocellular carcinoma (HCC) and other solid tumours with cancer spread already continue to be a therapeutic challenge that limits patient survival. Against this backdrop, laboratories in the NCCS are focused on developing new therapeutics to address these grim clinical situations.
Dr. Jian Cheng Hu’s laboratory focuses on uncovering the molecular mechanisms that underlie hyperactive Ras/RAS/MEK/ERK signalling-driven malignancies and drug resistance in targeted therapies with RAF/MEK inhibitors. Prof. William Hwang’s research focuses on expanding or replacing the role of hematopoietic and mesenchymal stromal cells to enhance hematopoietic recovery after high dose chemotherapy and transplantation, as well as for the treatment of autoimmune disease.
CANCER IMMUNOTHERAPYThe immune system identifies and destroys foreign “intruders” by discriminating “self” from “non-self.” Although cancer cells originate from patients’ own cells, they may be recognised and attacked by the immune system due to expression of tumourassociated antigens (TAA), which are not expressed by normal cells. These antigens may be viral antigens (e.g., Epstein-Barr virus) in virus-associated cancers, such as NPC, or “self” antigens, such as the family of cancer/testis antigens, the expression of which is restricted to germ cells and several cancers, including colorectal cancer, lung cancer, hepatobiliary cancers, and melanoma. A role for the immune system in cancer control has long been suggested, likely because of observations that there are significantly higher incidences of malignancies in immunocompromised patients, such as in organ transplant patients who receive immune-suppressive drugs. The rationale for tumour-specific cancer immunotherapy is thus to induce or boost such anti-TAA immune responses in these patients, with the aim to activate and expand TAA-specific cytotoxic T lymphocytes (CTLs) that kill tumour cells expressing the tumour antigen in question. Such therapeutic intervention could bein the form of a vaccination with TAA or TAA-pulsed antigenpresenting cells, such as dendritic cells (DC) derived from patient or donor peripheral blood monocytes, or through the adoptive transfer of ex vivo expanded antigen-specific cytotoxic T-lymphocytes.
The NCCS has been actively involved in the development of cellbased cancer immunotherapies since 2002. The Laboratory of Cell Therapy and Cancer Vaccine in NCCS, led by Assoc. Prof. Han Chong Toh, has achieved compelling results in their clinical trials of cellular immunotherapy strategies for treating solid tumours, such as colorectal cancer and NPC. The immunotherapy clinical trials that have been completed in the NCCS include:
With the encouraging results of this autologous EBV-specific cytotoxic T cell treatment for advanced NPC patients, Assoc. Prof. Toh and his team went on to establish a collaboration with a Singaporean biotechnology company, Tessa Therapeutics, and embarked on an ambitious project of conducting an international, multi-centre, randomised Phase III FDA IND clinical trial in similar NPC patients to further develop this treatment and bring it closer to approval. Besides Singapore, this trial also involves participating sites in Malaysia, Thailand,Taiwan, and the USA (following the successful clinical trial license application to Singapore’s Health Science Authority (HSA), Malaysia’s National Pharmaceutical Control Bureau [NPCB], the Thai and Taiwanese regulatory agencies and the US Food and Drug Administration [US FDA]). Assoc. Prof. Toh is the coordinating PI. Active recruitment of eligible patients was initiated in July 2014. In total, this trial has established 30 sites, including from the United States, Stanford; University of California, San Francisco; Massachusetts General Hospital; and City of Hope Cancer Center. This study aims to prove if the addition of T cells that can recognise and attack the virus proteins expressed on NPC can improve the survival of these patients over chemotherapy alone.
Another immunotherapy clinical trial currently underway in the NCCS is a First-in-Human Phase I Ad-sig-hMUC-1/ ecdCD40L cancer vaccine trial in collaboration with a US biotech company. This is an adenovirus vector-based cancer vaccine that aims to induce an anti-MUC-1 immune response in cancer patients whose tumours express the MUC-1 antigen, commonly expressed in epithelial cancers, such as lung, breast, colon, ovary, and prostate cancers. This trial was commenced in September 2014. Eighteen patients in the dose-escalation cohort have been recruited and an expansion cohort is ongoing. Comprehensive immune monitoring is also currently ongoing. Because the trial has shown that this cancer vaccine is safe with few side effects, the next objective is to show it can also have a significant anti-cancer benefit in a larger group of patients.
Assoc. Prof. Toh and his scientific team are also actively investigating other potential immunotherapeutic strategies. These include developing better gamma-delta T (γδT) cells for cancer treatment. In addition to conducting these benchto- bedside studies, the team is also actively conducting bedside-to-bench investigations, with efforts toward trying to discover biomarkers that predict the effectiveness of these immunotherapies for better patient selection as well as potential strategies to improve their efficacy. One such biomarker study is the identification of myeloid-derived suppressor cell levels as a powerful predictive marker for overall survival following EBVtargeting T cell therapy in advanced NPC patients.
Another immunotherapeutic strategy being actively pursued by several groups worldwide is the development of therapeutics aimed at modulating the immune–inhibitory microenvironment within tumours to make it favourable for the function of anti-tumour immune cells. One such strategy that has shown very promising results in several recent trials is the use of “checkpoint inhibitors.” Examples of these immune checkpoint inhibitors already approved by regulatory authorities such as the US FDA include Ipilimumab (anti-CTLA-4 antibody; produced by Medarex), Pembrolizumab (anti-PD-1 antibody; by MERCK), and Nivolumab (anti-PD-1 antibody; by Bristol-Myers Squibb). Led by the NCCS, a collaborative team of scientists from several institutions, including the Institute of Molecular and Cell Biology (IMCB), Singapore, and Duke-NUS Medical School, Singapore, as well as industrial partners, are studying the dynamic interactions between the immune microenvironment and selected solid tumours prevalent in this region, such as HCC and NPC, and investigating the possibility of developing novelnew therapeutics that target the tumour microenvironment to potentiate a more effective anti-tumour immune response. A major focus is to combine rational therapies to further improve the outcomes of single-agent immune checkpoint inhibitor therapy. This work is currently ongoing in pre-clinical animal models. To achieve this, the Toh laboratory collaborates actively with industry partners, including Big Pharma and emerging biotechnology companies.
Assoc. Prof. Toh now leads the VICTORY (Virus-Induced Cancer: Translational Oncology Research and immunology) consortium, an NMRC Large Collaborative Grant (LCG) that encompasses institutions across Singapore, including Duke-NUS-SingHealth; National Cancer Institute of Singapore, A*STAR; National Technological University; and multiple industry partners. This will build a more comprehensive and expansive programme of immunotherapy and translational immunology for virusinduced cancers, such as NPC, HCC, head and neck cancers, and cervical cancers. One of the key objectives is to develop better immunotherapies for such cancers to move into clinical trials.
Figure 1: Mature and antigen-pulsed dendritic cells ready to be harvested for use as a cancer vaccine.
Figure 2: Colonies of activated and proliferating Epstein-Barr virus (EBV)-specific T cells in culture.
DRUG DESIGN FOR HYPERACTIVE RAS/RAF/MEK/ERK SIGNALLING-DRIVEN CANCERSThe Ras/Raf/MEK/ERK (extracellular signal-regulated kinase) signalling cascade plays a central role in cellular proliferation, differentiation and survival. This signalling cascade is tightly and precisely controlled in normal cells, and its aberrant activation results in human cancers and other diseases. Table 1 shows the frequency of hyperactive Ras/Raf/MEK/ERK signalling across human cancers. Targeting this pathway for cancer therapy is one of hottest topics in current biomedical research, and is also the research goal of Dr. Jian Cheng Hu’s laboratory. Specifically, the laboratory is focusing on molecular mechanisms that govern hyperactive Ras/RAF/MEK/ERK signalling-driven cancer development and developing novel drugs against cancers with genetic alterations in this pathway.
DISTINCT REGULATORY MECHANISMS OF DIFFERENT RAF ISOFORMS AND THEIR ROLES IN CANCERSRaf kinase is a pivotal component of the Ras/Raf/MEK/ERK signalling cascade, which transmits a signal from GTP bound Ras to activate MEK by phosphorylation. Raf kinases consist of three family members: BRAF, CRAF, and ARAF. Genetic alterations that activate Ras/Raf/MEK/ERK signalling in cancers mainly occur through Raf kinase or upstream molecules. As such, Raf has been regarded as an ideal target for drug design.
Table 1: Hyperactive Ras/Raf/MEK/ERK Signalling in Cancers
Indeed, two Raf kinase inhibitors (vemurafenib and dabrafenib) have been developed and are used in the clinical treatment of late-stage BRAF (V600E)-positive cancer patients. Unfortunately, Raf kinase inhibitors do not impair but promote the growth of cancers that harbour Ras or RTK mutations. Mechanistic studies have shown that, in these cancers, wild-type Raf molecules are not inhibited but, paradoxically, activated by inhibitors through a dimerization-dependent mechanism (Hu J, et al., Cell, 2013; Lavoie and Therrien, Nat Rev Mol Cell Biol, 2015). Moreover, although BRAF is the dominant mutated member of Raf kinase in cancers, CRAF may also be a major player in Ras/RTK-mutated cancers. Unlike BRAF, CRAF forms a super complex with MEK, KSR (kinase suppressor of Ras), and other unknown proteins that are resistant to Raf or MEK inhibitors. These findings indicate that different Raf molecules are regulated through distinct mechanisms and contribute to the development of different types of cancers with similar molecular structures and an identical triggering of the MEK-ERK downstream pathway. An understanding of the regulatory mechanisms of these various complexing molecules will not only provide a conceptual framework for drug design against hyperactive Ras/Raf-induced cancers, but also propel the redesign of targeted therapies, and therefore improve clinical outcomes for cancer patients whose tumours harbour mutations in Ras, Raf, or RTKs.
MOLECULAR BASIS UNDERLYING BRAF(V600E)-INDUCED HAEMATOPOIETIC MALIGNANCYAmong the cancer-related mutagenesis spectrum of Raf kinases, BRAF(V600E) represents the most frequent mutation and accounts for >90% of cases. Moreover, BRAF(V600E) is enriched in specific cancer types, such as melanoma, papillary thyroid cancer, and colorectal carcinoma. Recent studies also identified BRAF(V600E) as a highly prevalent driver of hairy cell leukaemia and systemic histiocytosis; although, it is rare among all haematopoietic malignancies. Interestingly, somatic BRAF(V600E) mutations in haematopoietic malignancy occurs at the progenitor cell stage, whereas, in other types of cancers, it occurs during the terminal differentiated cell stage. Whereas the BRAF(V600E) mutation in haematopoietic stem cells induces hairy cell leukaemia, the same mutation in mature B cells or differentiated B cell progenitors is not associated with disease. The molecular basis underlying this phenomenon is still a puzzle, and its resolution will contribute much to improving the current treatment strategies for BRAF(V600E)-positive hairy cell leukaemia/systemic histiocytosis.
ENHANCING HAEMATOPOIETIC TRANSPLANTATION STRATEGIESProf. William Hwang’s research team focuses on expanding or replacing the role of haematopoietic and mesenchymal stromal cells to enhance haematopoietic recovery after high-dose chemotherapy and transplantation as well as for the treatment of autoimmune disease. The team has developed a small molecule that can expand blood stem cells effectively and that can significantly improve the outcome of haematopoietic blood stem cell transplants, especially cord blood transplants. Interestingly, this molecule appears to encourage the growth of the most primitive blood stem cells while suppressing the growth of leukaemia cells, suggesting a potential for use in accelerating haematopoietic recovery with simultaneous leukaemia control. In addition, Prof. Hwang’s team has developed a two-factor (2F) combination that can effectively treat graft versus host disease as well as autoimmune disease in mice. These technologies have been patented and the group is currently looking to bring both treatments to clinical trials. The laboratory has also recently identified a new role for the stromal microenvironment in the pathogenesis and disease progression of myelodysplastic syndromes, a pre-leukaemic disorder of the bone marrow. This could provide a basis for the introduction of new cancer drugs that target the stromal microenvironment and the means for identifying patient populations that may benefit from such therapy.
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