Leukemia Research Vol. 16, No. 1, pp. 47-50, 1992. 0145-2126/92 $5.00 + .00 Printed in Great Britain. Pergamon Press plc

GENE MUTATIONS IN MYELODYSPLASIA

A L L A N J A C O B S

University of Wales College of Medicine, Cardiff, U.K.

Abstract--The myelodysplastic syndrome is a paradigm of human preleukaemia. Normal haemo- poiesis is progressively displaced by an abnormal clone derived from a mutated stem cell. The initial mutation is unknown but its occurrence may be related to the overall load of random mutations which are a consequence of both intrinsic DNA defects and external mutagens. Evolution of the pathological population is marked by an increasing load of genetic lesions at the molecular and cytogenetic levels. Ras mutations can be detected in the blood of about 50% of MDS patients. Fms mutations are less common but these lesions can be found both in patients and in haematologically normal subjects who have previously received cytotoxic therapy suggesting that they can occur early in the preleukaemic process. Clonal haemopoiesis in the absence of either ras or fms mutations can occur in these subjects. The datasuggest the inability of mutant ras or fms genes alone to produce observable preleukaemic changes but that subjects with these mutations may be predisposed to future MDS. Ras mutations are a common accompaniment of a wide variety of malignancies and experimental transfection of the mutant gene can induce a malignant phenotype in cultured cells. There are many possible mechanisms for this transformation which may be relevant in a clinical context. Experimentally observed effects include a direct influence on the cell cycle, the induction of drug resistance and the stimulation of autocrine growth factor production. It may eventually be possible to define which gene mutations are important in conferring a malignant state, which determine phenotype and which are of incidental significance.

Key words: Myelodysplasia, ras, fins, preleukaemia.

I N T R O D U C T I O N

IN THE myelodysplastic syndrome (MDS) a clonal population of haemopoietic stem cells arising from an initial genetic insult progresses to a preleukaemic state and ultimately to overt acute myeloblastic leukaemia [1, 2]. The mechanism of progression from a trivial haematological aberration to overt leukaemia involves successive genetic changes resulting in ab- normal control of cell proliferation and differentia- tion. We are now beginning to define the molecular lesions in the genome that are associated with myelo- dysplasia and the development of leukaemia.

The number of possible target genes in the stem cell alone is enormous and our decision to investigate the role of ras and fms mutations was made on the following grounds. Ras mutations have been found in a wide variety of both animal and human malignancies [3]. These affect H-ras, N-ras and K-ras and are usually found at codons 12, 13 and 61. Their widespread association with malignancy, including acute myeloblastic leukaemia [4,5] suggests an im- portant role in the pathogenesis of neoplasia. In addition there is the accumulated evidence of the importance of the ras protein (p21) in signal trans-

47

duction and the control of cell proliferation, the ability of transfected mutant ras genes to confer a malignant phenotype in certain cultured cells and the potentiality for using biological techniques in vitro for assessing malignant transformation in the investi- gation of clinical material.

The investigation of point mutations in the fms gene was stimulated by recent work demonstrating differences between v-fms and c-fms, c-fms codes for a 150kD protein identified as the receptor for CSF-1. The extracellular ligand binding domain has immunoglobulin-like features and the intracellular domain has tyrosine kinase activity, v-frns, the transforming gene, has a number of point mutations compared to c-fms both in the extracellular and intra- cellular domains with a deletion of the C-terminal tail. Roussel et al. [6] studied the transforming activity of a number of c-frns constructs to determine which mutations were of pathological significance. While c-fms with a phenylalanine mutation at codon 969 had no transforming activity either in an NIH 3T3 focus formation assay or in cultures examined for the presence of anchorage-independent colonies, c-fins with a serine substitution at codon 301 in the extra- cellular domain, was transforming in both systems.

48 A. JACOBS

The presence of a phenylalanine 969 mutation greatly potentiated the transforming activity of the serine 301 mutant DNA. The tyrosine residue at codon 969 is thought to serve a negative regulatory function, loss of which releases the receptor from its control.

Mutant ras genes

We assessed mutational activation of H-ras, K-ras and N-ras in patients' marrow and blood cells [7]. The polymerase chain reaction was used to amplify sequences around the target codon and hybridization was carried out with wild type and mutant-specific synthetic oligonucleotide probes. Genomic DNA was transfected in NIH 3T3 focus forming assays and nude mouse tumourigenicity assays. Material from 75 cases of MDS has now been examined and the total number of mutations found was as follows:

SA RA RAEB CMML Total

No. of cases 16 18 10 31 75 No. with mutations 4 6 4 26 40 % mutations 25 33 40 84 53

It must be assumed that ras activation plays a part in malignant progression by conferring a selective growth advantage on the cells in which the mutations occur, and, possibly, by interfering with their differ- entiation programme. The presence of mutations in patients with the most benign forms of MDS, presumably at an early stage in the preleukaemic process, suggests that it may be an early event. However, there appears to be a high rate of leukaemic transformation in patients with mutant ras genes. In 39 of our patients with a ras mutation, 14 have progressed to acute leukaemia while only 3 out of 36 with no mutations have transformed during a two year follow-up. Present evidence suggests that ras mutations may be found both in early and in late stages of haemopoietic malignant progression. Amongst 70 patients treated for lymphoma by standard chemotherapy regimes between 1 and 13 years previously and with no residual disease or haematological abnormality, 9 were found to have mutant ras genes by PCR and hybridization--again suggesting that this can be an early lesion in the preleukaemic process [8].

In an initial attempt to determine the incidence of detectable mutant ras genes in the peripheral blood cells of normal subjects we detected mutations in 2 subjects out of 18 examined, 1 H12 Asp and the other H12 Val, which were not only easily detectable through hybridization in vitro but were also tumouri- genic in nude mice. One of the female subjects was heterozygous for RFLPs around the PGK locus on

the X chromosome and DNA from both polymorph and lymphocyte populations were shown to be mono- clonal [9]. These results suggest (a) a high incidence of ras mutations in a normal population, (b) the inability of mutant ras genes alone to produce observ- able preleukaemic changes, (c) the existence of subjects predisposed to future preleukaemic change.

Mutant fms genes

Sequences containing codons 301 and 969 were amplified by PCR from peripheral blood or marrow cells from 64 patients with MDS and 47 with AML, 4 of whom had been examined in an earlier MDS phase. The amplified material was screened by hybridization to mutant-specific oligonucleotide probes with the detection of two mutations at codon 301 and 14 mutations at codon 969. Mutations appear to occur in about 20% of patients with CMML and M4 AML but with a significantly lower frequency in other types of MDS. In two cases fins mutations were found in AML patients that were not present in an earlier MDS stage. In one case of RAEB a f ins mutation dis- appeared after transformation to AML [10].

In haematologically normal patients studied following cytotoxic therapy, 11 out of 70 were found to have a mutation at codon 969, three of whom also had a ras mutation [11]. This supports the suggestion that point mutations can be an early manifestation of the leukaemogenic process, giving rise to clonal haemopoiesis at an earlier stage than overt haemato- logical abnormality. The majority of codon 969 mutations showed a cysteine substitution. Confirma- tion of these mutations by direct sequencing is currently in progress.

The significance o f point mutations in M D S

The high frequency of these point mutations in patients with preleukaemia is not entirely surprising. They were found in all clinical types of the disorder, including the relatively benign groups and those on the verge of overt leukaemia. It was noticeable, however, that both ras and f m s mutations occurred more commonly in those patients whose disease had a monocytic phenotype, either chronic myelomonocytic leukaemia or acute myeloblastic leukaemia of FAB type M4. The reason for this is by no means clear, and we cannot be sure whether these mutations themselves induce differentiation along the mono- cytoid pathway, or whether cells already committed to monocytoid differentiation are unduly susceptible to the biological effects of the activated genes. The latter seems more probable, at least for ras. In mature B cells, ras mutation may induce terminal differentia- tion and plasma cell morphology in vitro [12].

We do not, of course, know what molecular lesions,

Gene mutations in myelodysplasia 49

are present in those patients with preleukaemic haemopoiesis who do not have either ras or fins mutations or whether these mutations can be initiat- ing events. Using the M2715 X chromosome probe [13], we investigated all female subjects in the group studied after chemotherapy for evidence of clonal haemopoiesis. Clonality was demonstrated in six out of seven women having either ras or frns mutations but, in addition, 3 women were found to have clonal haemopoiesis in the absence of detectable f ins or ras mutations, suggesting that in these cases at least another initiating lesion must have been present.

We can only speculate about the possible mech- anisms through which the mutant p21 ras contributes towards malignant transformation. The common transforming mutations reduce the protein's GTPase activity [3] with the result that they remain "switched on" for prolonged periods without the need for external stimulation. The consequences of this are not known but it is possible to contemplate some of the possibilities. One of the effects of ras protein is to induce expression of l o s [14, 15] and there is good evidence to suggest that the transformed phenotype produced by transfected mutant ras in vitro is de- pendent o n f o s expression [16]. Increased stimulation of the cell cycle at the G0/Gl phase might therefore be an immediate effect of activated ras genes. Ras is expressed at the G1/S phase and activates p60 cdc25 at G2/M. Although we can only speculate on the overall effect of mutant ras on cell cycle control we know that it is involved at a number of points and may be responsible for the haemopoietic cell cycle ab- normalities observed in MDS.

One of the more intriguing characteristics of ras- transformed cells in culture is their increased resist- ance to toxic drugs and chemicals. In rat liver epithelial cells this is associated with increased ex- pression of the m d r - 1 and the glutathione-S trans-

ferase genes [17]. Increased drug resistance and expression of the m d r - 1 gene has been observed in the haemopoietic cells of patients with myelodys- plastic syndromes and acute myeloblastic leukaemia before they have had any cytotoxic drug exposure [18] and it is tempting to suggest that it is this feature that leads to the selective proliferation of ras- transformed haemopoietic cells.

Griffin [19] has shown that mesothelioma cells transfected with mutant N-ras contain increased transcripts for a number of cytokines, including G- CSF, GM-CSF, IL-I~ and IL-6. If this were true for the transformed haemopoietic stem cells in our preleukaemic patients we can see how mutant ras genes might result in both autocrine and paracrine stimulation within the bone marrow. Many diverse mutations have already been described in different

types of malignancy. Which ones have special sig- nificance in leukaemogenesis remains to be seen.

R E F E R E N C E S

1. Jacobs A. (1987) Human preleukaemia: do we have a model? Br. J. Cancer 55, 1-5.

2. Oscier D. G. (1987) Myelodysplastic syndromes. Clin. Haemat. 1, 389-426.

3. Barbacid M. (1990) Ras oncogenes: their role in neo- plasia. Eur. J. Clin. Invest. 20, 225-235.

4. Janssen J. W. G., Steenvoorden A. C. M., Lyons J., Anger B., Bohlke J. U., Seliger H. & Bartram C. R. (1987) RAS gene mutations in acute and chronic myelocytic leukemias, chronic myeloproliferative dis- orders, and myelodysplastic syndromes. Proc. natn. Acad. Sci. U.S.A. 84, 9228-9232.

5. Farr C., Saiki R. K., Erlich H. A., McCormick F. & Marshall C. J. (1988) Analysis of ras gene mutations in acute myeloid leukaemia by polymerase chain reaction and oligonucleotide probes. Proc. natn. Acad. Sci. U.S.A. 85, 1692.

6. Roussel M. F., Downing J. R., Rettenmier C. W. & Sherr C. J. (1988) A point mutation in the extracellular domain of the human CSF-1 receptor (c-fins proto- oncogene product) activates its transforming potential. Cell 55, 979-988.

7. Padua R. A., Carter G., Hughes D., Gow J., Farr C., Oscier D., McCormick F. & Jacobs A. (1988) Ras mutations in myelodysplasia detected by amplification, oligonucleotide hybridisation and transformation. Leukaemia 2, 503--510.

8. Carter G., Hughes, D. C., Clark R. E., McCormick F., Jacobs A., Whittaker J. A. & Padua R. A. (1990) RAS mutations in patients following cytotoxic therapy for lymphoma. Oncogene 5, 411-416.

9. Jacobs A. Genetic lesions in preleukaemia. Leukaemia (in press.)

10. Ridge S. A., Worwood M., Oscier D., Jacobs A. & Padua R. A. (1990) FMS mutations in myelodysplastic, leukemic, and normal subjects. Proc. natn. Acad. Sci. U.S.A. 87, 1377-1380.

11. Jacobs A., Ridge S. A., Carter G., Hughes D. C., Clark R. E., Whittaker J. A., Padua R. A. & Cachia P. G. (1990) FMS and RAS mutations following cytotoxic therapy for lymphoma. Expl Hemat. 18, 648.

12. Seremetis S., Inghirami G., Ferrero D., Newcomb E. W., Knowles D. M., Dotto G. & Dalla-Favera R. (1989) Transformation and plasmacytoid differentia- tion of EBV-infected human B lymphoblasts by ras oncogenes. Science 243, 660-663.

13. Abrahamson G., Fraser N. J., Boyd Y., Craig I. & Wainscoat J. S. (1990) A highly informative X-chromo- some probe m27b can be used for the determination of tumour clonality. Br. J. Haemat. 74, 371-377.

14. Stacey D. W., Watson T., Kung H. & Curran T. (1987) Microinjection of transforming ras protein induces c- los expression. Mol. cell. Biol. 7 (1), 523-527.

15. Gauthier-Rouviere C., Fernandez A. & Lamb N. J. C. (1990) Ras-induced c-los expression and proliferation in living rat fibroblasts involves c-kinase activation and the serum response element pathway. E M B O J. 9 (1), 171-180.

16. Ledwith B. J., Manam S., Kraynak A. R., Nichols W.

50 A. JA¢OBS

W. & Bradley M. O. (1990)Anfisense-fos RNA causes partial reversion of the transformed phenotypes in- duced by the c-H-ras oncogene. Mol. cell. Biol. 10 (4), 1545-1555.

17. Burt R. K., Garfield S., Johnson K. & Thorgeirsson S. S. (1988) Transformation of rat liver epithelial cells with c-H-ras or v-ras causes expression of MDR-1, glutathione-S-transferase P and increased resistance to cytotoxic chemicals. Carcinogenesis 9, 2329-2332.

18. Holmes J., Jacobs A., Carter G., Janowska-Wieczorek A. & Padua R. A. (1989) Multidrug resistance in leukaemic cell lines, myelodysplastic syndromes and acute myeloblastic leukaemia. Br. J. Haemat. 72, 40--44.

19. Demetri G. D., Ernst T. J. & Griffin J. D. (1989) Transfection of a mutant N-ras oncogene into human mesenchymal cells induces aberrant expression of several cytokine genes by increasing mRNA stability. Blood 74, 193a.