Mechanisms of cancer therapy resistance and evolution

A PhD project for the 2022 doctoral clinical fellows programme with Dinis Calado (primary supervisor, Crick) and John Riches (Barts Cancer Institute/QMUL)

Project description

A significant proportion of lymphoma patients (~40%) are refractory or relapse to standard therapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone: R-CHOP). Still, more than 20 years after R-CHOP implementation, this therapy remains the “gold standard” notwithstanding numerous (>20) Phase-III clinical trials[1]. 

John Riches

Clinical Senior Lecturer & Group Leader, Barts Cancer Institute/QMUL

Fundamentally, we lack information on the biology of R-CHOP-resistant lymphoma cells responsible for patient relapse. Patient re-biopsy early after treatment represents a serious ethical challenge. The collection of diagnostic-relapse paired samples is not common practice and their study has so far failed to provide consistent relapse predictors. Also, patient derived xenografts ultimately lack context and are unsuitable for immune-microenvironment investigation. 

Our work at the Immunity and Cancer Laboratory at The Francis Crick Institute demonstrates feasibility in using mouse models of lymphoma for in-depth analysis of lymphoma samples at diagnosis, posttreatment and relapse[2-4]. To this aim we have successfully established a R-CHOP-like treatment in the mouse and have developed lymphoma mouse models of therapy relapse. To better understand resistance mechanisms, their prevalence and targetability we considered that studying a lymphoma in a single mouse, at diagnosis, posttreatment and relapse, is a requirement. We therefore optimized surgical procedures that allow partial tumour resection[4]. The expectation is that the knowledge gathered in the models complements human studies, providing a more complete understanding of why lymphoma patients fail R-CHOP treatment.

(PhD Year 1-2): Using mouse models of lymphoma the aim is to collect paired lymphoma samples at diagnosis, R-CHOP posttreatment and relapse. The study of diagnostic versus posttreatment lymphoma samples will be used to identify candidate mechanisms of resistance and to investigate whether those mechanisms are acquired during R-CHOP therapy or pre-programmed. Analysis of posttreatment versus relapsed lymphoma samples allows investigation on features associated with resistance at posttreatment that are retained in relapsed samples or instead lost during lymphoma evolution. Because mechanisms of resistance may be genetic, epigenetic and/or metabolic we will perform characterisation of lymphoma cells at the various stages of treatment using bulk, single cell RNAseq and ATAC-seq coupled with metabolic profiling and genomic sequencing. To investigate the immune microenvironment[5] we will apply spatial transcriptomics together with flow and mass-cytometry analysis.

(PhD Year 2-3): Perform inter-species (human/mouse) lymphoma comparisons to identify alterations that function as biomarkers of R-CHOP therapy failure/success and/or that allow to risk-stratify patients to the appropriate treatment. Building on our previous experience[2-4] we are in particular interested to investigate alterations that impact the activity of MYC, BCL2 and/or the NF-kB pathway, which have previously been associated with poor responses to R-CHOP. These analysis will be followed by the functional testing of the impact of candidate mechanism/s on outcome following R-CHOP treatment through the modification of lymphoma cells derived from tumours of the established mouse model systems in the laboratory using e.g. CRISPR-CAS technology. 

The project involves the following training skills: bulk and single cell genomics; metabolomics; bioinformatic analysis of high content data using R/Python; pathological analysis using immunohistochemistry, fluorescence and mass-cytometry; multicolour flow-cytometry; PET and ultrasound imaging techniques; genetic manipulation using CRISPR-CAS technology.

The partner institution for this project is Barts Cancer Institute/QMUL.
 

References

  1. Pfreundschuh, M., Schubert, J., Ziepert, M., Schmits, R., Mohren, M., Lengfelder, E., . . . German High-Grade Non-Hodgkin Lymphoma Study, G. (2008). Six versus eight cycles of bi-weekly CHOP-14 with or without rituximab in elderly patients with aggressive CD20+ B-cell lymphomas: a randomised controlled trial (RICOVER-60). Lancet Oncol 9: 105-116. PubMed abstract
  2. Calado, D. P., Zhang, B., Srinivasan, L., Sasaki, Y., Seagal, J., Unitt, C., . . . Rajewsky, K. (2010). Constitutive canonical NF-kappaB activation cooperates with disruption of BLIMP1 in the pathogenesis of activated B cell-like diffuse large cell lymphoma. Cancer Cell 18: 580-589. PubMed abstract
  3. Zhang, B., Calado, D. P., Wang, Z., Frohler, S., Kochert, K., Qian, Y., . . . Rajewsky, K. (2015). An oncogenic role for alternative NF-kappaB signaling in DLBCL revealed upon deregulated BCL6 expression. Cell Rep 11: 715-726. PubMed abstract
  4. Maybury, D. B., Y., S.-T., A., S. T. J., J., F. and P., C. D. ( 2021). Generation and surgical analysis of genetic mouse models to study NF-κB-driven pathogenesis of Diffuse Large B-Cell Lymphoma, in NF-κB transcription factors. Methods Mol Biol in press. PubMed abstract
  5. Riches, J. C., Davies, J. K., McClanahan, F., Fatah, R., Iqbal, S., Agrawal, S., . . . Gribben, J. G. (2013). T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production. Blood 121: 1612-1621. PubMed abstract