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Inhibition of the T-cell receptor signalling pathway for treatment of T-cell lymphoma

Periodic Reporting for period 2 - TITLY (Inhibition of the T-cell receptor signalling pathway for treatment of T-cell lymphoma)

Reporting period: 2016-11-01 to 2017-10-31

Non-Hodgkin lymphoma (NHL) is a form of cancer emerging from the transformation of a mature B- or T-cell lymphocyte. Clinically, NHL is usually characterized by lymph nodes and/or spleen enlargement, variable bone marrow or peripheral blood involvement and occasionally extra-nodal spreading of the disease. NHL is a highly heterogeneous disease with the last 2008 World Health Organization (WHO) classification encompassing more than 30 sub-entities. Heterogeneity of the disease translates into a highly variable prognosis for patients suffering from NHL ranging from 5-year overall survival (OS) of more than 90% in case of indolent B-cell lymphomas to less than 10% in the most aggressive subtypes of T-cell malignancies.

NHL is the 11th most common cancer in Europe, approaching 100,000 new cases diagnosed in 2012. Worldwide, NHL is the 10th most common cancer with the highest incidence in Northern America and the lowest in South Central Asia. This heterogeneity reflects potential underlying risk factors, quality of data collection and diagnostic screening methods. Among NHL, roughly 90% are of B-cell type (ie arising from the transformation of a mature B-cell lymphocyte) whereas 10% are of T-cell origin.

Recently, major advances have been made in the treatment of B-cell NHL from the understanding of signaling pathways crucially involved in the tumor cell survival and proliferation. On the contrary, pathophysiology of T-cell lymphomas is poorly understood and almost no therapeutic progress has been made for the last 20 years in the disease.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system is a bacterial immune system that has been modified for genome engineering. Largely due to its simplicity and adaptability, CRISPR has rapidly become one of the most popular approaches for genome engineering. CRISPR consists of two components: a single guide RNA (sgRNA) and a CRISPR-associated endonuclease (Cas9 for the streptococcus pyogenes endonuclease which is broadly used). The sgRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding and a user-defined ∼20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified. Thus, one can change the genomic target of Cas9 by simply changing the targeting sequence present in the sgRNA. When targeted to a locus defined by the sgRNA sequence, the Cas9 generates a double-stranded blunt cut ultimately leading to inactivating missense or frameshift mutations. Therefore, the CRISPR/Cas9 system was originally employed to “knock-out” target genes in various cell types and organisms.

Given the critical unmet medical needs for T-cell lymphoma treatment, we undertook in November 2015 an unbiased approach using whole-genome CRISPR/Cas9 screening in T-cell lymphoma cell lines to decipher essential signaling pathways for tumor cell survival and proliferation.

The ultimate goal of the project was to provide new therapeutic targets in the disease for rapid early phase clinical trial development.
The CRISPR/Cas9 technique enables inactivation of virtually any gene of the genome. By designing a specific 20 base-pair sequence complementary to the targeted DNA sequence, an RNA fragment named single-guide RNA (sgRNA) allows for recruiting the Cas9 endonuclease resulting in blunt ends DNA fragments and gene disruption (Figure 1).

During the outgoing phase of the action (from November 2015 to October 2016) that took place at the National Institute of Health (Bethesda, MD, USA), nine peripheral T-cell lymphoma (PTCL) cell lines have been engineered to express the Streptococcus pyogenes Cas9 enzyme : HuT78, HuT102, MyLa, HH and Sez4 (cutaneous PTCL); OCI-Ly12 and OCI-Ly13.2 (nodal PTCL); SUDHL1 and DEL (ALK+ anaplastic PTCL).
During the return phase at the Lyon Sud Hospital in France, whole genome CRISPR/Cas9 screens have been performed in all of these lines (Figure 2) and potential therapeutic targets have been individually confirmed.

Several genes essential for survival have been identified in keeping with the objectives of the action: BCL-xL, an anti-apoptotic gene, was found to be of utmost relevance for therapeutic purposes in PTCL, as well as CFLAR, a gene inhibiting the extrinsic apoptotic pathway in various cell types. Based on those pre-clinical data, new early phase trials using specific inhibitors are expected to be set up in the next few months. Many other genes of putative therapeutic importance have been also identified and are in patenting process.

Results have been presented at many national and international meetings (International Conference on Malignant Lymphoma, Lugano, June 2017; Lyon Center for Cancer Research, November 2017; Imagine Workshop, Necker Hospital, Paris, January 2018) reaching a broad audience of researchers, industrial partners, and patients' associations.
In conclusion, during the timeframe of the current project, whole-genome CRISPR/Cas9 screens have been performed in various PTCL cell types. To the best of our knowledge, no such whole-genome screens in this particular disease has been published so far. Results have identified new relevant targets that will pave the way for drugs development in the forthcoming years.

The work has led to major technical and scientific breakthroughs by providing a compendium of essential pathways in T-cell lymphoma and by allowing repurposing of drugs in current development for other malignancies. Regarding commercial impact, several genes with therapeutic potential are in the patenting process.

The current project funded by the European Union H2020 program allowed me to set up a new research group in Lyon as a principal investigator (Center for Cancer Research, to get a tenure-track position as an Associate Professor and to secure other research fundings (>100K€).

During the return phase of the action, whole-genome CRISPR/Cas9 screens have been implemented in my lab with the use of an onsite next-generation sequencer (Illumina NextSeq 500). Transfer of knowledge has been effective with numerous research teams now involved in collaborative projects both at the local and the national levels (collaborations with the International Center for Infectiology Research, Lyon, France; the Imagine Institute, Paris, France; Institut Cochin, Paris, France).

A large effort of knowledge dissemination has been performed towards the civil society and especially patients' associations (Association France Lymphome Espoir, Meeting in September 2017, Lyon, France), industrial partners (CALYM French Institut Carnot for the development of innovation, meeting in January 2018, Paris, France), and the medical research community (Lugano International meeting in June 2017, Switzerland).
Overview of the whole-genome screen steps
Schematic representation of the CRISPR/Cas9 system