Myeloproliferative neoplasm (MPN) is classified as the disorder of hematopoietic progenitor cells with various mutations being implicated in its pathogenesis, including JAK2, MPL, and CALR driver mutations. We focused on CALR, an ER chaperone involved in protein folding and calcium homeostasis, The CALR mutations are primarily observed in Exon 9, resulting in partial (CALR ins5) or complete (CALR del52) deletion of negatively charged amino acids present in the CALR WT tail. While both the CALR mutations activate TpoR mediated JAK/STAT signalling, they differ significantly in their phenotype and prognosis, hinting at differential regulation of downstream signalling cascades. Consistently, co-targeting PI3K-mTOR and JAK-STAT signalling through BEZ235 and Ruxolitinib, increased the efficacy of these drugs in mice models and in MPN progenitor cells. Therefore, we aimed to dissect the interplay between CALR mutations implicated in MPN and mTOR signalling to pave the way for potential future targeted therapeutics.

We utilized CALR overexpression systems (WT, Del52, Ins5) in HEK cells to investigate the regulation of mTOR (mTORC1 and mTORC2) signalling cascade through immunoblottings. As these mutations target the CALR tail, we created CALR WT truncation mutants to understand the extent of activation of mTOR signalling. Furthermore, we used inhibitors to assess any distinction between CALR mutations in the inhibition of mTOR cascade. We have compared the glucose uptake and cellular energetics through ATP levels in the cell between CALR WT and mutations. We also performed mass spectrometry studies, siRNA-based knockdowns, and overexpressed mTOR complex-specific protein overexpression to establish the molecular axis behind the observed phenotype.

We have established differential activation of mTORC2 but not mTORC1 by CALR mutants, with mTORC2 being significantly downregulated in CALR Del52. This further resulted in a shift in the lysosomal count, distribution, mTOR colocalization, and transcriptional activity of the TFE3 gene, a key component for lysosomal biogenesis and autophagy induction. Our mass spectrometry data reveals the potential interactors that play a role in the lysosomal distribution in the cells.

We have studied the effect of CALR mutants on different pathways and found that CALR ins5 mutant triggers high mTORC2 (S2481) activation compared to CALR WT or CALR del52. This selective activation of mTORC2 by CALR mutant and its downstream implications on lysosomal distribution and biogenesis is an interesting finding. This study highlighted the difference between CALR mutants del52 and Ins5 in differential modulation of mTOR pathway and its downstream impact on lysosomes.