TET2 Mutations

The TET1 gene was identified as a rare translocation partner with the mixed-lineage leukemia gene (MLL) in a fraction of patients with AML. Neither this gene, nor the other two genes it shares homology with, TET2 and TET3 , had previously been associated with malignancy. In the last year, TET2 was reported to be mutated at high frequency in several myeloid malignancies, including MDS. Using SNP-arrays, analysis of DNA samples from patients with MDS, MDS/MPN, and AML revealed recurrent acquired uniparental disomy and small deletions in chromosomal segment 4q24. The only open reading frame in the smallest commonly altered region was the TET2 gene and mutations of highly conserved codons in TET2 were identified in these patients. TET2 mutations were not limited to a single class of myeloid disorders. Many patients with MPN (∼10%) had TET2 mutations (often associated with loss of heterozygosity of 4q24) as did a large fraction of unselected patients with MDS (∼20%), chronic myelomonocytic leukemia (CMML) (∼30%), and secondary AML (∼25%), including those without obvious alterations of the 4q24 locus. Subsequent studies have confirmed that TET2 mutations are not exclusive of several genetic abnormalities and are found alongside PML-RAR , FLT3-ITD , JAK2 V617F , MPL W515L/K , KIT D816V , NRAS , and NPM1 mutations and in myeloid malignancies with no known mutations. Point mutations of TET1 or TET3 were not found in a large set of MPN samples and have not been reported in other myeloid malignancies to date.

No other recurring gene mutation has been identified in such a broad spectrum of myeloid neoplasms at such high frequency, which prompts the speculation that impairment of TET2 function promotes a common element in the pathogenesis of all of these diseases, namely the establishment or maintenance of clonal hematopoiesis. Mutations of this gene could not be the sole cause of the dysplasia seen in patients with MDS because patients with polycythemia vera (PV) carrying TET2 mutations have normal (albeit amplified) erythroid differentiation. Nor does TET2 seem to promote transformation to acute leukemia in isolation. In a study of 96 subjects with MDS, TET2 mutations were no more likely to be found in those with high risk disease nor were any new mutations seen during disease progression. In another study, TET2 mutations were found more frequently in low and intermediate-1 risk groups.

Instead, it appears that impairment of TET2 function may provide a survival advantage to early hematopoietic progenitors without profound effects on differentiation or proliferation. Single colony assays of CD34 ⁺ cells from the bone marrow of patients with JAK2 V617F and TET2 mutations support this view. A subset of more primitive CD34 ⁺ CD38 ⁻ cells from these patients often carried TET2 mutations without associated JAK2 mutations. The proportion of TET2 -only mutant cells was greater in this CD34 ⁺ CD38 ⁻ group than in more mature CD34 ⁺ CD38 ⁺ cells. In the five subjects studied, no cells carrying the JAK2 V617F mutation were found to have wild-type TET2 . CD34+ stem cells from two subjects with TET2 mutated/ JAK2 V617F positive neoplasms could repopulate the bone marrow of sub-lethally irradiated NOD-SCID mice, whereas cells from three subjects with the JAK2 mutation and wild-type TET2 could not. Fifteen weeks after engraftment, single colony assays of surviving cells revealed that the population of TET2 mutated, JAK2 wild-type cells had grown at the expense of those cells carrying mutations in both genes. This finding suggests that the TET2 mutation might have preceded the JAK2 V617F mutation in these subjects and that it resides in a more primitive hematopoietic stem cell.

Evidence for mutations in TET2 as an early event in myeloid disorders is not universal, however. Saint-Martin and colleagues studied four family cohorts with a strong inherited predisposition for MPNs and AML. These subjects did not carry germline JAK2 mutations, but often acquired them when they presented with myeloid malignancies. Many of the affected members of these cohorts developed TET2 mutations, but several others did not, and no two related subjects acquired the same TET2 mutation. In one subject with JAK2 V617F / TET2 -mutant PV for which several bone marrow samples were available, it was noted that the subject had mostly JAK2 V617F -positive clones that were TET2 negative at an early stage of the disease. The remaining clones were either completely wild-type or contained the JAK2 V617F mutation and a single TET2 mutation. When the subject progressed to acute leukemia, almost all bone marrow mononuclear cells contained biallelic TET2 mutations in addition to JAK2 V617F . These findings demonstrate that TET2 mutations can be either primary or secondary events and are consistent with impaired TET2 function creating a selective growth advantage for disease stem cells.

Less is known about the role of TET2 mutations in MDS. In a study of 96 subjects with MDS, Kosmider and colleagues found mutation of TET2 to be a positive prognostic factor independent of IPSS risk group. There was no difference in clinical markers at the time of presentation in the mutated compared with the non-mutated group. In contrast, other groups have found that patients with AML or CMML that carry TET2 mutations have decreased overall survival. It is not clear why TET2 mutations appear to be protective in MDS, but are unfavorable in more proliferative myeloid diseases. It is possible that patients who have MDS with TET2 mutations as the basis of their disease lack other, less favorable genetic lesions, and hence have better outcomes. But for the subset of patients with more aggressive diseases, the lack of TET2 function could enhance the effect of the additional mutations they have acquired or impart a relative resistance to treatment. Studies with a much larger number of subjects are needed to better understand the prognostic implications of TET2 mutations.

The mechanism through which TET2 mutations promote diverse myeloid malignancies is not well understood. However, a recent study on the function of its homolog TET1 suggests that TET2 may affect epigenetic modulation of gene expression. TET1 encodes a 2-oxoglutarate and Fe(II)-dependent enzyme that catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). This modified base was found to comprise roughly 5% of all CpG islands in mouse embryonic stem cells. The authors speculate that 5-hmC could be a chemical intermediate in the demethylation of cytosine residues, either by spontaneous or enzyme-mediated conversion. Alternatively, DNA methyltransferases may not recognize 5-hmC, and therefore, may not methylate cytosines on newly synthesized DNA strands complementary to regions with these epigenetic marks. The result would be a net loss of cytosine methylation mediated by TET proteins. MDS and AML are disorders whose pathogenesis involves hypermethylation of several tumor suppressor genes, and are diseases for which inhibitors of DNA methyltransferases have shown efficacy. It is intriguing to have identified a frequently mutated gene whose likely mechanism of action could explain the basis for these observations. Of particular interest will be the analysis of whether TET2 -mutation status predicts response to therapy with the hypomethylating agents 5-azacytidine and decitabine.

ASXL1 Mutations

The ASXL1 gene is another example of a gene that has recently been found to carry mutations in a variety of myeloid disorders including MDS. This gene encodes a chromatin-binding protein that functions as a ligand-dependent coactivator of the retinoic acid receptor. It regulates the response to retinoic acid in a cell-type specific manner and is thought to mediate its effects through a direct interaction with the histone acyltransferase NCOA1 (also known as SRC-1 ) or the histone demethylase LSD1 . ASXL1 is located in 20q11, but is proximal to the 20q common deleted region in patients with MDS.

ASXL1 was identified as a myeloid disease gene from aCGH experiments analyzing germline and CD34 ⁺ -cell DNA from a small set of patients with MDS/AML. One individual had a small, atypical deletion on chromosome 20 that encompassed the ASXL1 locus. Subsequent sequencing identified mutations of this gene in 16% of subjects with MDS/AML and in 43% with CMML. Most of these samples were studied to see if they carried mutations in several other MDS associated genes, including HRAS , KRAS , NRAS , RUNX1 , NFIA , CTNNB1 , and TET2 . In this small sample set, mutations in RUNX1 and TET2 were identified in subjects with MDS and these did not coexist with ASXL1 mutations. In CMML cases, RUNX1 and TET2 mutations were not exclusive of ASXL1 mutations. In contrast, ASXL1 mutations were found in several MPN samples, but none of these were from subjects with PV and none carried the JAK2 V617F mutation. This finding suggests that loss of ASXL1 activity might replace the pathogenicity of the constitutively activated JAK2 oncogene in a subset of JAK2 wild-type myeloproliferative diseases. As was the case with CMML samples, TET2 mutations were not exclusive of ASXL1 mutations in these MPNs. Mutations in ASXL1 were also identified in 20% of a group of subjects with AML, most of which had normal cytogenetics. Although nearly half of these subjects had NPM1 mutations, ASXL1 and NPM1 mutations were epistatic. In this study, the investigators suggest that this mutual exclusivity could mean that ASXL1 and NPM1 contribute to the same pathogenic pathway. The alternative interpretation is that these genes represent completely independent mechanisms of transformation because many of the ASXL1 mutated AML cases arose from chronic myeloid diseases, whereas most NPM1 -mutated cases did not.

Exactly how ASXL1 contributes to the development of MDS and other myeloid disorders is not clear. ASXL1 deficient mice do not have a dramatic myeloerythroid phenotype or gross changes in their stem cell compartment. The higher incidence of ASXL1 mutations in CMML and AML compared with MDS and its mutual exclusivity with JAK2 in MPNs prompts the speculation that ASXL1 mutations may drive cell proliferation with few effects on morphology or differentiation. Finally, the clinical implications of ASXL1 mutations in patients with MDS are not yet known.

CBL Mutations

The CBL gene encodes an E3 ubiquitin ligase and contains a tyrosine kinase binding domain. It negatively regulates tyrosine kinase activity by promoting ubiquitination of these proteins, leading to their degradation. Using the same approach that led to the discovery of TET2 mutations, mutations of the CBL gene were found in a large fraction of patients with myeloid disorders who were noted to have acquired uniparental disomy of 11q. Most of these patients had CMML or atypical chronic myelocytic leukemia (aCML), but CBL mutations were found in a few patients with refractory anemia with excess blasts. In several CBL mutants without 11q UPD, a second inactivating mutation in CBL was identified. Although some mutations appear to cause loss of function, other evidence indicates that CBL may function as an oncogene. Mutations in CBL occur almost exclusively in a short segment of the protein coding region and maintain the reading frame of the gene. The mutated protein product has impaired ubiquitin ligase activity and can impair the function of wild-type CBL in a dominant negative fashion. Expression of mutant CBL in 3T3 cells enhances their ability to form colonies in agar and tumors in nude mice. In this sense, CBL appears to be an oncogene and not a tumor suppressor. The answer is that it may be a little of each. Mice deficient for cbl have an expanded hematopoietic progenitor pool and some degree of hypersensitivity to cytokine signaling, but do not develop MDS or MPN. When a mutant CBL gene is inserted into hematopoietic cells from these mice, they show dramatic hypersensitivity to cytokine stimulation and proliferate much more. It appears that CBL mutants gain the ability to promote tyrosine kinase signaling that is separate from their loss of ubiquitin ligase activity, possibly through dominant negative inhibition of its homolog CBLB .

CBL mutations have previously been identified in rare cases of de novo AML, and are most frequently associated with core-binding factor leukemias. Most CBL mutant samples from patients with AML are negative for mutations in FLT3 , KIT , NPM1 , CEBPA , and RAS genes, implying that loss of CBL function can substitute for the growth advantage conveyed by these oncogenic mutations more commonly associated with AML. CBL mutations are also found at high frequency (17%) in cases of juvenile myelomonocytic leukemia (JMML). These mutations are exclusive of RAS and PTPN11 mutations, which are associated with 25% to 35% of JMML cases.

Although CBL mutations have been found in patients with MDS, it is primarily associated with the monocytic myeloproliferative disorders JMML, CMML, and aCML. Its mechanism of action and lack of association with other pro-proliferative oncogene mutations suggest that mutation of CBL has an effect comparable to that caused by constitutive activation of tyrosine kinases. It remains to be seen whether CBL mutations play a role in lower risk MDS or whether loss of this protein function confers a worse prognosis in this disease.

Receptor Tyrosine Kinase and RAS Pathway Mutations

Activating mutations in the RAS family of oncogenes were among the first frequently recurring point mutations identified in patients with MDS. They are present in roughly 15% of MDS cases and are associated with an increased risk for progression to AML. Various missense mutations in codon 12 of NRAS are the most common RAS mutations in MDS, with mutations at other sites or in KRAS occurring at lower frequency. Like CBL mutations, oncogenic RAS mutations are more common in patients with CMML (∼50%) and JMML (∼30%).

RAS proteins are mediators of signal pathways downstream of receptor tyrosine kinases, such as those that mediate cytokine sensitivity in hematopoietic cells. These include receptors for thrombopoietin, erythropoietin, stem cell factor, Flt-3 ligand, platelet-derived growth factors, several interleukins, insulin and insulin-like growth factors, and the colony stimulating factors. Some of these receptors have been found to have activating mutations in advanced cases of MDS or CMML, including FLT3 , CSF1R (aka c-FMS ), and KIT , although these are more commonly associated with AML. Other RTK-RAS pathway genes mutated in myeloid disorders include BRAF and PTPN11 , both of which are rare in MDS.

TP53 Mutations

Mutations of the critical cell-cycle checkpoint gene TP53 are among the most common genetic abnormalities in malignancies. In MDS, TP53 mutations occur in 10% to 15% of patients, but this frequency can be higher in those with prior exposure to alkylating agents or radiation. Inactivating mutations of TP53 are associated with advanced disease, complex karyotype, and resistance to treatment; all of which portend a poor prognosis. The presence of TP53 mutations may even be an independent risk factor after accounting for IPSS risk score. As the sensitivity of molecular diagnostics improve, it will be feasible to determine if patients carry TP53 mutations in diseased subclones at low frequency. This treatment resistant TP53 -mutant population may drive progression of disease and expand after therapy even in patients who otherwise appear to have a good prognostic profile.

RUNX1 Mutations

RUNX1 is a member of the core-binding factor family of proteins and an important regulator of normal hematopoiesis. Like TET2 , RUNX1 abnormalities are found in many myeloid disorders. In MDS, mutations are present in 7% to 15% of cases of de novo disease, but appear to be more frequent in therapy-related MDS. Patients who have MDS with RUNX1 mutations are more likely to have advanced disease and carry a poor prognosis.

The RUNX1 protein contains two primary domains important for its function: a proximal Runt DNA binding domain and a distal activation domain. Mutations are found throughout the length of the gene in patients with de novo MDS, but tend to cluster in the DNA binding domain in patients with AML and therapy-related MDS. Heterozygous Runt domain mutations can act as dominant negatives, severely limiting RUNX1 activity, and may be partially responsible for the poorer outcome of patients with therapy-related disease.

Manipulation of RUNX1 in mice has helped explain how acquired RUNX1 mutations could lead to MDS and AML. Complete RUNX1 knockout results in embryonic lethality without evidence of definitive hematopoiesis. However, selective RUNX1 excision in adult animals does not block hematopoiesis. Instead, these mice show lymphoid defects, expansion of the myeloid progenitor pool, inefficient platelet production, and extramedullary hematopoiesis, but not progression to AML. In contrast, mice transplanted with bone marrow cells overexpressing RUNX1 with a Runt domain mutation developed increasing bone marrow blast counts, splenomegaly, and often died of an AML-like disease. Mice transplanted with cells expressing RUNX1 with a more distal frameshift mutation instead developed a more MDS-like phenotype with marked erythroid dysplasia and pancytopenia with a lower rate of progression to AML.

Further evidence of the role of RUNX1 in myelodysplasia and progression to AML comes from the rare autosomal dominant disease known as the familial platelet disorder with propensity to acute leukemia (FPD). Affected persons with FPD often have an MDS-like disease characterized by low number of platelets that may function abnormally. About 35% of patients with FPD will develop AML, often by acquiring a second RUNX1 mutation in their remaining wild-type allele.

In the mouse models and human cases of congenital RUNX1 mutations, progression of disease occurs only after a long latency and likely requires cooperating mutations. In patients with MDS and patients with AML arising from MDS, RUNX1 mutations have been associated with activating mutations in the receptor tyrosine kinase-RAS pathway and the presence of chromosome 7 deletions, which suggests that these abnormalities are complementary, each driving a distinct step in the pathogenesis of more advanced disease.

JAK2 Mutations

Activating mutations of JAK2 are the defining genetic lesion of PV and are found in roughly half of patients with essential thrombocythemia (ET) or primary myelofibrosis. In MDS, the mutation rate is much lower with the JAK2 V617F mutation reported in approximately 5% of unselected patients with MDS. This frequency is higher in a subset of patients. More than half of patients with refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) carry the JAK2 V617F mutation and a smaller number contain activating mutations of MPL . These patients typically have low risk disease and rarely progress to acute leukemia. This mutation frequency is comparable to that seen in essential thrombocytosis, prompting the speculation that some cases of RARS-T could be considered to be the frustrated expression of a myeloproliferative disorder and not always a subcategory of MDS. The alternative view is that the acquisition of a JAK2 V617F mutation in the setting of RARS leads to thrombocytosis, but patients retain the risk of progression associated with RARS.

There remains a population of JAK2 V617F positive patients who have MDS without ringed sideroblasts. Whether these patients have an MDS/MPN overlap syndrome or simply MDS is less clear. Analysis of a 5q- MDS cohort demonstrated the JAK2 mutation in 6% of these patients. Those positive for JAK2 V617F had higher peripheral white blood cell counts, a trend toward a higher platelet count, but no excess blasts in their bone marrow. One patient sample was subject to clonal analysis, identifying the 5q- abnormality in 91% of metaphases. However, single colony assays revealed the JAK2 V617F mutation in closer to 50% of cells, which suggests that the JAK2 mutation developed in a subclone that had already acquired the 5q- deletion. In this genetic background, constitutive JAK2 activity may provide a clonal advantage without being able to drive the proliferation of terminally differentiated cells seen in PV and ET. It also suggests that targeting JAK2 activity in patients who have MDS with JAK2 V617F mutations might do little to ameliorate the risk of subsequent disease progression.