Shp2 regulates leukemic stem cell frequency in MLL-rearranged acute myeloid leukemia

Shp2 is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. It contains two SH2 domains, a PTP domain, and a carboxyl-terminal region with tyrosine phosphorylation sites. The Shp2 PTP activity is autoinhibited by its N-SH2 domain. Shp2 is activated by docking to specific tyrosine phosphorylated sites via SH2 domains or by gain-of-function (GOF) mutations that disrupt the autoinhibition. GOF Shp2 mutations are most frequently detected in juvenile myelomonocytic leukemia (JMML), representing 35% of cases. Animal model studies using conditional knockin of GOF Shp2^E76K or Shp2^D61G in the hematopoietic compartments have shown that these Shp2 mutants cause JMML-like myeloproliferative disease (MPD), albeit after ~6 months of latency (1,2). The leukemogenic activity of Shp2 mutants is cell autonomous, indicating that these Ptpn11 mutants are driver oncogenes. These Shp2 mutant-expressing mice have hyperactive hematopoietic stem cells (HSCs) with a decrease of Linage^-/Sca-1⁺/c-Kit⁺ (LSK) cells in the bone marrow and an increase of LSK cells in the spleen (1,2). Besides JMML, GOF PTPN11 mutations have also been found in other hematologic malignancies and in solid tumors but at lower rates, including acute myeloid leukemia (AML) (3). In pediatric AML, PTPN11 mutations are most prevalent in the French-American-British (FAB)-M5 subtype, which is monoblastic leukemia. FAB-M4 (acute myelomonoblastic leukemia) and FAB-M5 morphological subtypes of AML correlate with MLL-rearrangement (4). The prevalence of PTPN11 mutations in a particular AML subtype that displays similar morphologically feature as the PTPN11-driven JMML suggests that PTPN11 mutations are not merely passenger mutations. However, this was not proven experimentally. Since PTPN11 mutations are present in a fraction of AML cells from each patient, these mutations do not appear to be the primary oncogenic events leading to leukemogenesis (3). Therefore, the role of Shp2 mutations in AML was not clear.

In a new report published online in Leukemia (5), Chen et al. identified six cases of GOF PTPN11 mutations among 91 pretreatment AML samples (6.6% mutation rate). These six mutations (E76G, T73I, E76Q, D61Y, F71L, and E76G) are located in exon 3 that encodes the N-SH2 domain. Mutations in these residues are highly predictive of GOF that result in constitutively active Shp2. Based on saturation analysis of cancer genes in AML, these PTPN11 mutations are predicted to be functionally important. Other genetic alterations in DNMT3α, NPM1, WT1, CBF, and MLL were found in these PTPN11 mutated AML samples. This is consistent with the previous observation that PTPN11 mutations did not associated with any specific genetic lesions in pediatric AML (3).

To begin analyzing the potential function of PTPN11 mutations in AML, Chen and colleagues determined if a mutant Shp2 could affect the MLL oncofusion in murine bone marrow progenitor cell transformation. Shp2^E76K and MLL-AF9 were chosen as models of Shp2 and MLL mutations. Shp2^E76K is perhaps the strongest activating Shp2 mutant in exon 3. MLL-AF9 rearrangement is present in approximately 50% of pediatric AML cases (4). Shp2^E76K did not show significant transformation activity in their experiments but increased MLL-AF9 transformed colonies in methylcellulose cultures. Intravenous injection of Shp2^E76K-, MLL-AF9-, and MLL-AF9/Shp2^E76K-retrovirus transduced lin^-/kit⁺ bone marrow cells into lethally irradiated recipient mice showed that Shp2^E76K alone did not cause leukemia in 150 days, whereas it reduced the MLL-AF9-mediated disease latency by 2 folds. To determine if Shp2^E76K affects leukemic stem cells, Chen and colleagues performed limiting dilution assay by injecting primary leukemic cells from diseased mice into secondary recipients. The data showed a 5-fold increase in leukemia stem cell frequency in MLL-AF9/Shp2^E76K induced leukemia.

The presence of Shp2^E76K did not influence MLL-AF9 target genes Hoxa9 and Meis1 expression, suggesting that Shp2^E76K did not exert its effect on leukemogenesis by affecting MLL-AF9 transcription activity. Instead, hematopoietic progenitor cells transduced with MLL-AF9 together with Shp2^E76K showed IL-3 hypersensitivity in methylcellulose colony growth and Erk activation. Hypersensitivity to GM-CSF or IL-3 is the hallmark of biological consequences of activated Shp2 mutants in hematopoietic progenitor cells. Furthermore, cells transduced with MLL-AF9/Shp2^E76K had elevated Mcl1 and were resistance to Mcl1 inhibitors as compared with cells transduced with MLL-AF9 alone.

These findings provide evidence of functionality of PTPN11 mutations in MLL-rearranged AML by demonstrating that Shp2^E76K increased leukemic stem cell frequency. Consistent with the observation that PTPN11 mutations did not associated with particular genetic lesions in pediatric AML, the Shp2 mutant does not act directly on MLL-AF9. It will be interesting to determine if Shp2 mutants also affect leukemic stem cell frequency in cooperation with other genetic alterations that co-exist with PTPN11 mutations in AML.

Acknowledgements

Funding: J Wu was supported by NIH R01CA178456.

Footnote

Conflicts of Interest: The author has no conflicts of interest to declare.

References

1. Chan G, Kalaitzidis D, Usenko T, et al. Leukemogenic Ptpn11 causes fatal myeloproliferative disorder via cell-autonomous effects on multiple stages of hematopoiesis. Blood 2009;113:4414-24. [PubMed]
2. Xu D, Liu X, Yu WM, et al. Non-lineage/stage-restricted effects of a gain-of-function mutation in tyrosine phosphatase Ptpn11 (Shp2) on malignant transformation of hematopoietic cells. J Exp Med 2011;208:1977-88. [PubMed]
3. Tartaglia M, Martinelli S, Iavarone I, et al. Somatic PTPN11 mutations in childhood acute myeloid leukaemia. Br J Haematol 2005;129:333-9. [PubMed]
4. Balgobind BV, Zwaan CM, Pieters R, et al. The heterogeneity of pediatric MLL-rearranged acute myeloid leukemia. Leukemia 2011;25:1239-48. [PubMed]
5. Chen L, Chen W, Mysliwski M, et al. Mutated Ptpn11 alters leukemic stem cell frequency and reduces the sensitivity of acute myeloid leukemia cells to Mcl1 inhibition. Leukemia 2015. [Epub ahead of print]. [PubMed]