Anaplastic Lymphoma Kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) is a T-cell non-Hodgkin lymphoma characterised by a chromosomal rearrangement [t(2;5) (p23;q25)] involving the ALK and NPM1 genes on chromosomes 2 and 5 respectively. This translocation generates the NPM-ALK fusion protein, a hyperactive tyrosine kinase that is the driving oncogenic event in ALCL. Single-agent use of tyrosine kinase inhibitors (TKI) to inhibit ALK is a conventional second-line treatment and has been trialled for upfront use in combination with the ALCL99 therapeutic backbone. However, an extended duration of treatment is required as cessation of therapy can lead to rapid relapse although this is often salvageable with re-administration of the same or an alternative ALK TKI. In addition, for some patients, resistance can develop, and this tends to happen soon after starting treatment. Hence, whilst ALK TKIs hold much promise for the treatment of ALCL, there remain clinical issues that need to be addressed. One approach would be the introduction of a second targeted drug that has a different mechanism of action to be used in combination with ALK TKIs, which may potentiate these effects and could improve clinical outcomes.

To identify alternative drug targets for ALK-positive ALCL in particular, those that may, when inhibited, have synergistic effects with ALK TKIs, cell lines were employed to investigate oncogenic activity of NPM-ALK in the nucleus of ALCL. Whilst NPM-ALK is known to be located in the cytoplasm where it drives the activity of several signalling pathways, its nuclear location and functions remain largely unexplored. Initially, I showed that NPM-ALK is in the chromatin fraction within the nucleus and that it binds to DNA, although likely not directly, but rather through a protein complex. Furthermore, by conducting NPM-ALK: DNA crosslinking followed by CUT&RUN- sequencing I showed that NPM-ALK, albeit indirectly, drives the expression of an RNA methyltransferase, N6-adenosine-methyltransferase 70 kDa subunit (METTL3). This enzyme catalyses N6-methyladenosine (m6A), the most abundant mRNA modification, through a complex with METTL14 and WTAP. Whilst METTL3 has been described as both an oncogene and a tumour suppressor gene in different cancers respectively, its expression and link to NPM-ALK activity in ALCL led me to investigate its oncogenic activities.

Following exposure of cells to ALK TKIs or NPM-ALK knockdown, thereby inhibiting METTL3 activity, I showed by nucleoside mass spectrometry that cells contained reduced levels of m6A in polyA+ captured RNA. Furthermore, METTL3 was shown to be required for cell growth following shRNA-specific knockdown. A similar effect was observed by employing a novel, potent and selective first-in-class METTL3 inhibitor (METTL3i). Furthermore, inhibition of METTL3 activity with either the METTL3i, or knockdown of METTL3 combined with NPM-ALK inhibition showed significant additive activity in reducing cell viability. To validate this activity in a more clinically relevant model, a patient-derived xenograft (PDX) of ALCL adapted to culture in vitro was exposed to ALK and METTL3 inhibitors or METTL3 knockdown. Again, the combination of ALK and METTL3 inhibition showed a reduction in cell survival with additive effects. The mechanism for the observed reduction in cell viability was investigated further showing that the 3rd generation ALK TKI Lorlatinib led to cellular senescence, but that in addition to METTL3i, these cells underwent apoptosis. These data suggest that not only will the combination of ALK TKI and METTL3 inhibitor decrease viability, but it may also prevent residual senescent cells from reseeding tumour growth. Of interest, an additional PDX-derived cell line from an ALK TKI-resistant (Crizotinib) patient tumour was sensitive to METTL3 inhibition and showed near synergistic effects with the second-generation ALK TKI, Brigatinib.

Subsequently, to identify alternative potential treatment options, drugs that affect epigenetic activities in the cell were investigated for their ability to impact ALCL cell survival in vitro. ALCL cell lines were exposed to METTL3i in combination with an ‘epigenetic drug’ library of 281 compounds that directly or indirectly affect epigenetic targets for 48 hours, and cell survival was monitored by a resazurin-based assay. This drug screen showed that METTL3i could potentiate several classes of ‘epigenetic drugs’ such as a G9a/GLP histone lysine methyltransferase inhibitor that was ineffective in reducing ALCL cell viability when used as a single agent.

In conclusion, METTL3 is a novel downstream effector of NPM-ALK activity that acts in an oncogenic capacity to drive cell survival. Hence, inhibition of METTL3 represents a novel treatment approach that has additive activity with ALK TKIs and other ‘epigenetic drugs’ and may therefore prevent the development of ALK TKI resistance and long-term use of ALK inhibitors in the future.