Investigating the Efficacy of Ofatumumab and Rituximab in Abrogating the Survival of NOTCH1 Mutated Chronic Lymphocytic Leukaemic (CLL) Cells
Authors: Middleton Odette , McCaig Alison , Leach Mike , Michie Alison , Wheadon Helen 
Published: November 2019

Abstract
The acquisition of cytogenetic abnormalities and genetic mutations contribute towards the heterogeneity of CLL progression. Patients harbouring NOTCH1 mutations (NOTCH1MUT) are identified as a poor prognostic subset, with the mutation conferring increased cell survival and proliferation. A retrospective study of the CLL8 clinical trial identified that NOTCH1MUT patients did not benefit from the addition of rituximab (RTX) to standard first line therapy. While trial results investigating the efficacy of combining ofatumumab (OFA) and chlorambucil suggested an initial benefit for both NOTCH1 wild type (NOTCH1WT) and NOTCH1MUT patients, progression free survival of NOTCH1MUT patients was not significantly enhanced by the inclusion of OFA. This study prompted us to analyse how NOTCH1 mutations impact on OFA/RTX-mediated antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We also assessed how proliferating CLL cells respond to OFA in NOTCH1WT and NOTCH1MUTsamples at the level of CD20 expression, ability to induce CDC, and changes in gene expression profiles (GEPs).
Of 89 CLL patients screened for NOTCH1 mutations, 8 had NOTCH1 mutations in the absence of 11q and 17p deletions. We assessed the ability of these cells to undergo ADCC and CDC following OFA or RTX treatment in vitro, and detected no significant difference in response between NOTCH1MUT and NOTCH1WTCLL patients. However in both CLL cohorts we did demonstrate that OFA more efficiently induced both ADCC and CDC compared with RTX.
Interaction of CLL cells with the microenvironment of lymphoid organs promotes survival and proliferation, and generates cytoprotective signals that must be overcome to provide a curative therapy for CLL. To compare how NOTCH1MUT and NOTCH1WT CLL patients respond to OFA treatment in vitro in pro-proliferative conditions, three different culture systems were used: plastic (media alone), NTL (stromal cells) and NTL-CD154/IL-4 (stromal cells expressing CD40L supplemented with IL-4). Flow cytometric analyses revealed differential regulation of CD20 expression levels between NOTCH1MUT and NOTCH1WT samples, with 40% of NOTCH1WT CLL samples up-regulating CD20 surface expression on NTL-CD154/IL-4, with no change observed in NOTCH1MUT samples. Microenvironmental conditions, in the presence/absence of OFA impacted the OFA-mediated CDC response between NOTCH1WT and NOTCH1MUT patients. Indeed, post NTL-CD154/IL-4 co-culture the OFA-CDC response was significantly higher in NOTCH1WT samples, with a mean increase in CDC of 36.5% (p=0.005; n=5), compared to CLL cells on plastic, while an elevation in CDC did not occur in NOTCH1MUT CLL patients. Conversely when NOTCH1MUTpatients were pre- incubated with OFA on NTL-CD154/IL-4 they produced significantly higher levels of CDC (37.5%; p=0.02; n=5) when compared to pre-incubation with OFA on plastic.
GEPs of fresh NOTCH1WT and NOTCH1MUT CLL samples revealed that NOTCH1MUT samples express significantly higher levels of DELTEX and lower levels of HES5, TP73, RRAD and CCNE2. Co-culture of both CLL cohorts on NTL-CD154/IL-4 resulted in a decrease in NOTCH1 levels, and other components of the Notch signalling pathway - HES1, LFNG and ADAM10. While both CLL cohorts up-regulated ROR2, FOS and JUN expression upon NTL-CD154/IL-4 co-culture, this was higher in NOTCH1MUT CLL cells. Interestingly, ROR2, FOS and JUN have been linked with Ca2+ channel activity and Ca2+ signalling, together with DAPK1 and RRAD, which were also deregulated in NOTCH1MUT CLL samples.
In conclusion, our studies indicate that the OFA-CDC response to microenvironmental stimulation cannot be entirely attributed to CD20 expression levels. Reduced OFA-CDC activity in NOTCH1MUT samples may be due to deregulation of genes involved in Ca2+ signalling, highlighting the role of CD20 as a store-operated Ca2+ channel.

Disclosures
Middleton: GlaxoSmithKline UK Ltd: Research Funding. Leach:GlaxoSmithKline UK Ltd: Research Funding. Michie:GlaxoSmithKline UK Ltd: Research Funding. Wheadon:GlaxoSmithKline UK Ltd: Research Funding.