The Lymphoma research programme in the NCCS is spearheaded by Prof. Soon Thye Lim alongside Dr. Choon Kiat Ong from the Lymphoma Genomics Translational Laboratory. This programme is built upon a strong collaborative foundation between oncologists, pathologists, and scientists, working synergistically toward a common goal of understanding the disease and finding better treatments for our patients. Taking on the “bench-to-bedside” approach, our research programme strives to shed light on genomic and molecular mechanisms that serve as drivers for disease initiation, promotion, and progression in the various subtypes of lymphoma (Figure 1). The discoveries from the basic research stage of this “bench-to-bedside” workflow lay the foundations upon which future diagnostic and therapeutic studies in the pre-clinical and clinical stages will arise.
Figure 1. “Bench-to-Bedside” approach of our programme
LEADERS IN GENOMIC SEQUENCING
One of the team’s fortes is in genomic sequencing and analysis, and we arecurrently leading the whole-genomic sequencing of T- and NK-cell lymphoma in the International Cancer Genomic Consortium (ICGC). This platform has allowed us to study the complexities of these cancers. Sequencing works performed in other lymphoma subtypes have also enabled us to understand the influence of germline and somatic mutations on each subtype. Through these efforts, we have identified novel therapeutic targets and markers that are currently in development for clinicalapplications.
For example, following our genome-wide association study (GWAS) for diffuse large B-cell lymphoma (DLBCL), we performed a GWAS for natural killer/T-cell lymphoma (NKTL), which showed multiple gene aberrations associated with NKTL susceptibility. The 51 single nucleotide polymorphisms (SNPs) identified were mapped to the class II MHC region on chromosome 6, with the most significantly associated SNP (rs9277378) for NKTL located in a gene coding for an antigen receptor, thereby linking antigen processing and presentation to disease pathogenesis (Figure 2).
Figure 2. Association of SNPs within the broad HLA region of naturalkiller/T-cell lymphoma.
Lymphoma is a very heterogeneous disease and accurate diagnosis is an important aspect of improving patient outcomes. It is particularly important for T- and NK-cell lymphoma, as they are less studied and limited information is available. To improve the diagnosis of this group of lymphomas, we have collaborated with the National Institute of Health to perform microarray gene expression profiling, attempting to identify the expression signature for each subtype. Besides being able to identify and reclassify some of the PTCL-NOS (Peripheral T-cell lymphoma not otherwise specified) cases, the gene expression profiling further identified two major molecular subgroups in the remaining PTCL-NOS cases that were characterised by ahigh expression of either GATA3 (33%; 40/121) or TBX21 (49%; 59/121). The GATA3 subgroup was significantly associated with poor overall survival (P = 0.01). The high expression of a cytotoxic gene-signature within the TBX21 subgroup also showed poor clinical outcome (P = 0.05). In angioimmunoblastic T-cell lymphoma (AITL), high expression of several signatures in the tumour microenvironment was significantly associated with outcome, with a combined prognostic score predictive of survival in an independent cohort (P = 0.004). This contributes to the growing wealth of knowledge in terms of PTCL characterisation, and we are currently extrapolating these findings onto alternative platforms for clinical implementation and commercialisation of a diagnostic system.
Besides NKTL, we are also leading the research on monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), a fatal subtype of T-cell lymphoma with limited treatment options. We identified frequent alterations in the Janus kinase/signal transducers and activators of transcription (JAK–STAT) and G-protein-coupled receptor (GPCR) signalling pathways in patients with MEITL. We further demonstrated that these alterations are targetable by inhibitors of both the JAK–STAT and mitogen activated protein kinase–extracellular signal regulatedkinase (MEK–ERK) transduction pathways, indicating potential therapeutic strategies for this neoplasm. Importantly, to the best of our knowledge, we have established most probably the world’s first patient-derived xenograft (PDX) models for MEITL for the preclinical study of these inhibitors, hoping to bring these therapeutic options to patients in the near future. This study demonstrated our capability to translate basic discoveries to preclinical models and toward clinical applications.
To effectively bring findings from the basic discoveries to pre-clinical studies, we have established an inventory of Patient Derived Xenografts (PDX) models for several other subtypes of lymphoma including DLBCL, NKTL, and PTCL, in addition to MEITL (Figure 3). These PDX models are relevant for in vivo therapeutic drug testing and are highly sought-after by many pharmaceutical companies. To make PDXs relevant to immunotherapy, we are currently generating humanised mouse models. For the same purpose, we have also established a syngeneic mouse model to characterise some of the novel therapeutic targets identified in our basic discovery research.
Our effective cross-island and regional collaborations are enhanced by the established Singapore Lymphoma Study group and the Asian Lymphoma Study group, respectively. In this programme, various scientists and clinicians from different departments here at the NCCS lend their expertise and knowledge to the many different facets of our research, boosting its robustness.
For instance, support from Prof. Balram Chowbay, with his expertise in pharmacology, is used to further characterise the pharmacokinetics and pharmacodynamics of candidate drugs shortlisted from our ongoing study involving novel JAK3 inhibitors. At the same time, the influence pharmacogenomics has on the efficacy and bioavailability of these drugs across the different ethnic groups will be investigated in future earlyphase clinical trials.
Collaborating with other laboratories in the NCCS could also provide a viable option to further our understanding of various disease subtypes. Dr. Jiancheng Hu leads the laboratory of Cancer Signalling. His team specialises in cancer signalling pathways including the MEK–ERK cascade. In our previous whole-exome sequencing study, this particular pathway was shown to contribute to the pathogenesis of the MEITL.
Figure 3. Development of patient-derived xenograft (PDX) animal models has allowed us to conduct various studies, including drug testing and the characterisation of novel therapeutic targets.
Figure 4. Mechanism of activation of the nuclear factor-κB (NF-κB)pathway in activated B cell-like–diffuse large B-cell lymphoma (ABC-DLBCL). Mutations in four proteins: cluster of differentiation 79 A and B (CD79A/B), myeloid differentiation primary response 88 (MYD88), caspase recruitment domain family member 11 (CARD11) and tumour necrosis factor alpha induced protein 3 (TNFAIP3) are implicated in the B cell receptor-dependent and independent activation of the NF-κB pathway. The signal transducer and activator of transcription 3 (STAT3) signaling pathway is indirectly modulated by phosphoinositol-3- kinase (PI3K) via NF-kB target genes interleukin 10 (IL-10) and IL-6. Negative crosstalk between PI3K and Bruton non-receptor protein-tyrosine kinase (BTK) results in a rebound activation of p-BTK and p-AKT upon inhibition of PI3K and BTK, respectively. BTK inhibitor, ibrutinib, with PI3Kα/δ inhibitor, copanlisib, offers a promising combination therapy to prevent tumour survival.
Furthermore, we showed that an oncogenic mutation in the Gα subunit of the G-protein causes an upregulation in ERK phosphorylation, which drives cancer progression. Dr. Hu’s specialty in the MEK–ERK signalling pathway offers a potentialcollaboration to fully decipher the mechanisms of aberrant MEK–ERK signalling, and could lead to discoveries of new treatment strategies and novel therapeutic targets.
We also team up with many cancer specialists and scientists from local institutions: Dr. Yuh Shan Lee, Dr. Nicholas Grigoropoulos, and Dr. Chandramouli Nagarajan from the Singapore General Hospital (SGH), Prof. Wee Joo Chng and A/Prof. Siok Bian Ng from the National University Cancer Institute Singapore, and Dr. Chiea Chuen Khor of the Genome Institute of Singapore. These partnerships run the gamut from performing basic science research to conducting clinical trials. Internationally, we have partnered with notable collaborators from leading cancer institutes from China, Korea, and Hong Kong.
Our programme has garnered the support of various industry partners, which has created many new opportunities for research.
In collaboration with Bayer Pharmaceuticals, we have discovered that simultaneous blocking of PI3Kα and PI3Kδ dramatically enhances the anti-tumour profile in activated B cell-like–diffuse large B-cell lymphoma (ABC-DLBCL) models compared with selective inhibition of PI3Kδ, PI3Kα, or Bruton non-receptor protein-tyrosine kinase (BTK). The anti-tumour activity was associated with the suppression of p-Akt and blocking NF-κB activation driven by CD79mut, CARD11mut, TNFAIP3mut, or MYD88mut (Figure 4). Inhibition of PI3Kα/δ resulted in tumour regression in an ibrutinib-resistant CD79BWT/MYD88mut patientderived ABC-DLBCL model. Furthermore, the rebound activations of BTK and AKT were identified as a mechanism limiting a robust response from CD79Bmut-ABC-DLBCL to PI3K and BTK inhibitor monotherapies. A combination of ibrutinib with the PI3Kα/δ inhibitor copanlisib produced a sustained complete response in vivo in CD79Bmut/MYD88mut ABC-DLBCL models. Based on these findings, a tripartite (Bayer, Janssen and NCCS) clinical trial in DLBCL is currently being proposed, demonstrating the effectiveness of our translational research programme.
Our earlier works in identifying novel therapeutic targets in NKTL have discovered frequent JAK3 activating mutations in this Asian-prevalent disease. This discovery led to a collaborative study with Principia Biopharma to characterise a novel JAK3-specific inhibitor, PRN371, for clinical application. PRN371 effectively suppresses NKTL cell proliferation and induces apoptosis through abrogation of JAK3-STAT signalling. Moreover, the activity of PRN371 has a more durable inhibition on JAK3compared with tofacitinib in vitro, leading to significant tumour growth inhibition in a NKTL xenograft model harbouring a JAK3 activating mutation. These findings provide a novel therapeutic approach for the treatment of NKTL. Building on these studies, we are currently screening and testing novel JAK3-specific inhibitors in collaboration with SYNthesis Pte Ltd, an Australianbased medicinal biochemical company with expertise in JAK inhibitor design. This project is supported by an initial funding from National Health Innovation Centre Singapore, and we hope our effort in characterising these compounds will lead to clinical trials, benefiting these patients.
Our team of clinician-scientists and researchers are involved in a wide spectrum of clinical trials, ranging from early-phase studies to international, randomised, phase III studies. In conjunction in Servier, we ran a phase I study of a novel BCL2 inhibitor in relapsed and refractory B-cell lymphomas. We also recently completed an investigator-initiated phase I study of selinexor in combination with ICE (ifosfamide, carboplatin, etoposide) in relapsed and refractory T-cell lymphomas. We have two ongoing investigator-initiated phase II clinical trials for patients with relapsed/refractory DLBCL that we are conducting with SGH; ibrutinib with RICE (rituximab, ifosfamide, carboplatin, etoposide) for transplant eligible and lenalidomide with RGDP (rituximab, gemcitabine, dexamethasone, cisplatin)for transplant-ineligible DLBCL patients. Together with LYSA, the French lymphoma research cooperative group, we are part of an international, randomised phase III study comparing Romidepsin-CHOP versus CHOP in the first-line treatment of T-cell lymphomas. We are also running various other pharmaceutical-sponsored and investigator-initiated studies in the setting of relapse or refractory B- (including primary central nervous system lymphoma) and T-cell lymphomas with Bayer, Janssen, Roche, and Bristol-Myers Squibb, Pte. Ltd.
AWARDS & ACHIEVEMENTS
Over the years, we have managed to clinch several notable accolades. Recently, our team was awarded the American Association for Cancer Research (AACR) Team Science Award, for our work in furthering the knowledge of Asian-prevalent cancers while contributing to the progress of cancer detection, treatment and prevention. Members from the laboratory have also been invited to speak and present their work at multiple local and international conferences, providing another platform for them to share their knowledge with the scientific community. Our work has also been published in numerous high-impact journals, such as Leukemia, Nat Genet, andLancet Oncol.
FUTURE WORKSIn the years to come, we will be extending our genome-wide association studies to PTCL and AITL, with hopes to uncover the genomic profile and characteristics of these diseases. The laboratory will also focus on elucidating the various mechanisms involved in refractory and relapsed B-cell lymphomas, paying particular attention to DLBCL.
This knowledge will provide the necessary edge that is required to fulfil the unmet clinical need of drug resistance, a tricky task that has complicated the successful treatment of cancer. Greater insight into these diseases would thus allow for improved clinical outcomes and survival rates for our patients.
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