N-glycosylation is an essential pathway in CALR-mutant MPN

Calreticulin (CALR) is an endoplasmic reticulum (ER) chaperone involved in protein quality control and other cellular processes. The occurrence of somatic mutations in the ER chaperone CALR are frequent and are susceptible to initiating disease in myeloproliferative neoplasms (MPN). Mutant CALR (CALR^mut) and myeloproliferative leukemia protein (MPL) are known to bind pathogenically in CALR^mut-induced MPN. However, there is lesser understanding of this mechanism leading to a challenge for the development of clonally selective therapeutic agents. The MPN Hub has previously reported on the mechanism of CALR 52-bp deletion (CALR^del52) binding and activation of the MPL signal.

During the 63rd American Society of Hematology (ASH) Annual Meeting and Exposition, Anna Marneth¹ from Harvard Medical School, Boston, US, presented findings from a whole genome sequencing analysis exploring the genetic dependencies in oncogenesis driven by CALR mutations. An important basis for this study was previous findings of a dependency between N-glycosylation side of MPL and mutant CALR-MPL interaction. The key findings are summarized here.

Aims and methods

• Whole genome sequencing with pro-B murine cell line dependent on interleukin-3 (IL-3) (Ba-F3) using CRISPR knockout screen in CALR^del52 MPL-expressing hematopoietic cells compared with empty vector (EV) control cells, to identify genes needed for the growth of cytokine-independent transformed CALR^del52.
• Validation studies using single gene CRISPR Cas9 to confirm the findings from whole genome sequencing.
• Pharmacological screen was done to investigate clinical implications in pathways identified from previous CRISPR analyses.
• Ribonucleic acid (RNA)-sequencing data from platelets of patients with MPN were used to demonstrate the relevancy of N-glycosylation pathways in human MPN.

Results

Whole-genome sequencing: N-glycan biosynthesis and protein secretion pathways essential for CALR^del52 transformation

• The findings have shown that seven among 10 most depleted genes were involved in protein glycosylation and essential for the growth of CALR^del52 in the absence of IL-3.
  • Gene enrichment analyses also confirmed that N-glycan biosynthesis is the most significantly depleted pathway in CALR^del52 in the absence of IL-3 compared with EV or CALR^del52 in the presence of IL-3 (a false discovery rate [FDR] q value of < 0.001).
  • Other significant pathways were unfolded protein response, and the protein secretion pathways (with FDR q values of 0.014, and 0.025, respectively).

• A secondary CRISPR pooled screen for 250 genes was done to confirm these pathways and the results supported the finding that N-glycan biosynthesis was the most significantly differentially depleted pathway in CALR^del52-transformed cells.
  • In addition, MPL, JAK2, and CALR were significantly depleted in the absence of IL-3.
• 70% of genes in both primary and secondary CRISPR screen were linked to N-glycosylation.
• Four of the seven top hit genes encode proteins involved in the enzymatic activity of dolichol-phosphate mannose synthase (DPM1, DPM2, DPM3, and MPDU1), an enzyme essential for protein N-glycosylation.
  • These observations support previous studies showing the association between N-glycosylation sites on MPL and the lectin-binding sites on CALR^del52 for CALR^mut-driven oncogenesis.
• Proteins involved in N-glycan biosynthesis and essential for CALR^del52 transformation were DOLPP1, SRD5A3, DOLK, ALG14, ALG1, ALG2, ALG11, RFT1, GPI, MPI, PMM2, DPM1, DPM2, DPM3, and MPDU1.
  • As DPM2 was the most significant gene in the whole-genome sequencing, it was further evaluated in the validation study.

Validation study: DPM2 knockout abrogates CALR^del52 transformation

• Increased cell death, reduced p-STAT5, and decreased MPL cell-surface levels, when DPM2 is knocked out (DPM2 ko) demonstrated that DPM2 was essential for CALR^del52-mediated transformation.
• Interestingly, all cells including EV non-targeting guide (NTG), EV DPM2 ko, CALR^del52 NTG, and CALR^del52 DPM2 ko cells enriched with IL-3 were not affected by lack of DPM2.
  • However, IL-3 removal led to increased growth of a sub-clone of non-edited DPM2 wild-type (DPM2^WT) CALR^del52 cells, supporting the critical role of DPM2 for the survival of CALR^del52 cells.

Pharmacological screen: Inhibition of N-glycosylation pathway preferentially targets CALR^del52

• This screening investigated pathways significantly depleted in the CRISPR analyses and included 70 compounds.
• All compounds that target N-glycosylation pathway inhibited the growth of CALR^del52 cells in the absence of IL-3 compared to the growth of EV cells in presence of IL-3.

• CALR^del52 cells treated with 2-deoxyglucose (2-DG) 1 or 10 µg, a mannose mimetic that inhibits the incorporation of mannose into N-glycan biosynthesis, in the absence of IL-3 showed a delayed growth compared with EV cells treated in the presence of IL-3.
• 2-DG also reduced MPL cell surface levels in CALR^del52 cells in the absence of IL-3.

• Treatment with 2-DG in MxCre CALR^del52 mice showed a reduced platelet count (p < 0.0001) and megakaryocyte/erythroid progenitor (MEP) frequency (p = 0.03) compared with vehicle, indicating that CALR^del52 was sensitive to this compound in in vivo settings.

• RNA sequencing showed that hallmark apoptosis was significantly upregulated in MxCre CALR^del52 mice treated with 2-DG compared to CALR^del52 mice treated with vehicle (FDR q value = 0.17).

• To further validate that CALR^del52 cells were more sensitive to 2-DG in vivo, a competitive bone marrow (BM) transplantation experiment was carried out using CD45.2 UBC-GFP MxCre CALR^del52 knock-in and CD45.1 mice. Six-week treatment with 2-DG produced significant reduction in platelet counts (p = 0.005), and platelet chimerism (p < 0.001) compared with vehicle. In addition, BM chimerism in the long-term hematopoietic stem cells, which are known to have disease-initiating properties, was significantly reduced.

RNA sequencing in patients with MPN: N-glycan pathways upregulated in CALR^del52 platelets

• For this investigation, RNA sequencing of CALR-mutant platelets from patients with CALR-mutant essential thrombocythemia (ET) or myelofibrosis (MF) compared with platelets from healthy controls. Indeed, pathways involved in N-glycosylation, such as fructose and mannose metabolism, and trimming in the ER and calnexin CALR cycle, were found to be significantly upregulated in platelets from CALR-mutant patients versus controls, emphasizing the relevance of these findings to human MPN.

Conclusion

These genetic, pharmacologically focused analyses identified the N-glycosylation biosynthesis pathway as essential for oncogenesis driven by CALR mutation. Inhibiting this pathway by DPM2 knockout or treatment with 2-DG eliminated the growth of CALR-mutant cells in the absence of IL-3. In vivo analysis has also shown that the inhibition of N-glycosylation has a potential to normalize key characteristics of MPN, e.g., platelet counts, and selectively targets CALR^del52 over wild-type cells. This study may have further therapeutic implications through inhibiting N-glycosylation alone or in combination with other agents to advance the development of clonally selective therapeutic approaches.