WO2001065994A2

WO2001065994A2 - Method of and kit for assessing responsiveness of cancer patients to antifolate chemotherapy

Info

Publication number: WO2001065994A2
Application number: PCT/IL2001/000212
Authority: WO
Inventors: Yehuda Assaraf; Statvit Drori
Original assignee: Technion Research And Development Foundation Ltd.
Priority date: 2000-03-06
Filing date: 2001-03-06
Publication date: 2001-09-13
Also published as: WO2001065994A3; AU2001240998A1

Abstract

A method for the assessment of the responsiveness of a cancer patient to antifolate-containing chemotherapy, the method is effected by searching for a mutation or mutations in a reduced folate carrier (RFC) gene in cells derived from the patient.

Claims

67 leucovorin growth requirement (Table 6). Altogether, these results were suggestive of an altered RFC-mediated transport, as is further described herein.

Table 6 below summarizes the antifolate growth inhibition and folate growth requirement in parental human CEM leukemia cells and their GW70 subline. Antifolate activity is presented as IC₅₀, the antifolate concentration (GW1843 and MTX, nM) resulting in 50 % cell growth inhibition, after 72 hours drug exposure. Folate growth requirement is presented as EC₅₀, folate concentration (folic acid and leucovorin, nM) resulting in 50 % of maximal control cell growth.

TABLE 6 Antifolate growth inhibition and folate growth requirement in parental human CEM leukemia cells and their GW70 subline

a - IC50, antifo

Results are the mean ± S.D. of 4-6 independent experiments. b - EC50, folic acid or leucovorin concentration (nM) necessary to support 50% of maximal growth of control cultures.

Table 7 below summarizes the kinetic parameters of folic acid and MTX transport in CEM cells and the GW 1843 -resistant sublines. 68

TABLE 7

Kinetic parameters of MTX and folic acid transport in parental CEM cells and their GW 1843 - resistant sublines"

a - The kinetic parameters Km and Vmax for [ H] MTX and [ H] folic acid influx were obtained from Lineweaver-Burk plots of initial (3 min) uptakes rates performed at extracellular (anti)folate concentrations of 1-200 M; b - Results are the mean S.D. of four separate experiments performed in duplicates.

Transport studies, kinetic transport parameters and affinities for folic acid and MTX in CEM, GW70 and GW70/LF cells:

Transport analysis was conducted by monitoring radioactive MTX or folic acid uptake into cells in suspension. [³H]MTX influx in GW70, as compared to CEM cells, was decreased >10-fold (0.3±0.1 vs. 3.2±0.7 pmol/min/10^ cells; n=12); this was accompanied by a consistent reduction in the steady-state MTX levels (Figure 7a). These changes were associated with a 3 -fold in the transport Km for MTX and a 2.5 -fold decrease in the transport V_max (Table 7). In contrast, GW70 cells displayed a 10-fold decrease in the transport K_m for folic acid, while retaining the parental V_maχ for this folate (Table 7). Consequently, and consistent with the 2-fold decrease in the folic acid growth requirement, as is described hereinabove (Figure 6c and Table 6), GW70, as is compared to their parental CEM cells, exhibited a >3-fold increase in both the influx (0.19±0.05 vs. 0.66±0.17 pmol/min/10⁷ cells, 69 respectively; n=8) and steady-state transport levels of folic acid, under conditions, in which folic acid reduction was blocked by trimetrexate (Figure 7b).

The impaired antifolate transport via RFC in GW70 made transport kinetic measurements difficult. To promote RFC overexpression as recently shown (Jansen et al, 1998), GW70 cells were gradually adapted to grow on low folic acid concentrations, resulting in the establishment of GW70/LF cells, which required only sub-nanomolar leucovorin concentrations for their growth. Indeed, GW70/LF cells displayed 5-fold RFC gene amplification (Figure 8a), consequent overexpression ofthe 3.1 kb (native) and 2.1 kb (truncated) RFC mRNAs (Figure 8b), thus resulting in a prominent overproduction of the native ~80 kDa and truncated 43 kDa RFC proteins (Figure 8c). This was associated with a 21 -fold and 15-fold increase in the transport V_max for MTX and folic acid, respectively, in GW70/LF cells as compared to their parental cells (Table 7). This carrier overexpression in GW70/LF cells facilitated the detection of a marked increase in the transport affinity not only for folic acid (Table 7-) but also for leucovorin and 5-methyltetrahydrofolate and for DDATHF (Table 8-). In contrast, the transport affinity for GWl 843U89 and ZD1694 was notably decreased in GW70/LF cells (Table 8-).

Table 8 below summarizes the affinities, presented as

(μM) (anti)folate concentration eliciting 50 % inhibition of [³H]MTX influx, the latter presented at an extracellular concentration of 5 μM) of RFC from CEM-7A cells and antifolate-resistant cells, for various folate cofactors and folate analogs. 70

TABLE 8

Affinities of RFC from CEM-7A and antifolate-resistant cells for various folate cofactors and foi ate analogs⁰

a - The affinities of RFC for folate and antifolate substrates are given as the (anti) folate

3 concentration (M) eliciting 50% inhibition of [ H] MTX influx where the latter was at an extracellular concentration of 5 M; b - Results are the mean of S.D. of 3-4 separate experiments.

To characterize the RFC mutation(s) which may underlie this defective-transport based, antifolate resistance, DNA from GW70 and GW70/LF cells was subjected to the PCR-SSCP assay. GW70 (Figures 9a and 9c, lane b) and GW70/LF cells (Figures 9a and 9c, lane c) displayed an identical electrophoretic mobility pattern in exon 2, which was drastically different from that observed with parental CEM cells (Figures 9a and 9c, lane a). Sequence analysis revealed that both GW70 and GW70/LF cells contained in exon 2 three identical mutations, including a G to C, G to A, and G to T at nucleotide positions 179, 227, and 331, respectively (see Table 4 , above). These mutations resulted in the following amino acid substitutions: Val 29 to Leu, Glu 45 to Lys, and Ser 46 to He, respectively (Table 4 -, above). Additionally, whereas CEM (Figures 9b and 9d, lane a) and GW70 cells (Figures 9b and 9d, lane b) had a normal electrophoretic mobility pattern for exon 3 of RFC, 71

GW70/LF cells (Figures 9b and 9d, lane c) displayed a markedly altered pattern. Sequencing identified a G to C mutation at nucleotide 356 in exon 3, resulting in a substitution of His 88 for Asp (Table 4-, above). Furthermore, whereas parental CEM cells were heterozygous, thus containing two RFC species, one with Arg 27 (G at nucleotide 174) and another with His 27 (A at nucleotide 174), GW70 cells lost this heterozygosity and therefore contained only the Arg 27 allele. This loss of heterozygosity was consistent with the genomic rearrangements observed in the Southern analysis with DNA from both GW70 and GW70/LF cells when compared to parental CEM cells (lanes b, c vs. lane a in Figure 8 a).

Table 9 below summarizes the intracellular folate pools in parental CEM cells and their GW-resistant sublines in parental CEM cells and their GWl 843 -resistant sublines.

TABLE 9

Intracellular reduced folate pools in parental CEM cells and their antifolate-resistant sublines

a - Intracellular reduced folate levels (pmol/mg protein) values represent the means SEM from six separate determinations; b - Cells grown In the presence of 2 nM folic acid; c - Cells grown In the presence of 2.3 M folic acid; d - Data presented are derived from Jansen et al., 1998. 72

Thus, as shown below in Table 10, GW70 cells displayed a high level resistance to various antifolate anticancer drugs which use RFC as the main uptake route. In contrast, GW70 cells were hypersensitive to lipid-soluble antifolates including trimetraxate which do not use the RFC for their entry.

TABLE 10

The growth inhibitory effects of antifolates on human CEM cells and their GW1843-resistant sublines"

a - Data presented are IC50 (nM) values obtained after 72h drug exposure; b - Cells grown in the presence of 2.3 M folic acid; c - Not determined

Genomic PCR-SSCP assay screening tumor specimens: The applicability of the genomic PCR-SSCP assay for the screening of RFC mutations in tumor specimens was further explored. Thus, DNA from a total of 25 B-precursor and T-cell childhood acute lymphoblastic leukemia (ALL) specimens (the vast majority of which were derived at diagnosis) were screened. One DNA specimen obtained at diagnosis from a B-precursor ALL patient (termed Ml) which relapsed 15 month later, displayed an altered electrophoretic mobility pattern in exon 2 (Figures 10a and 10b, lane b, marked also by asterisk). Sequencing revealed that this patient contained a G to C mutation at nucleotide 261, which resulted in a substitution of His 56 for Asp in the first predicted loop (LI) of RFC, and at the same time contained a G to A shift at nucleotide 174 in the same exon, and was thus homozygous for 73

His 27 (Table 4, above). Interestingly, SSCP analysis of 30 DNA samples from both healthy individuals and leukemia patients identified three equally distributed polymorphic groups at nucleotide position 174: one third of the individuals were homozygous for G174, thus containing Arg 27 (Figures 1 la - 1 Id, arrowhead), the other third was homozygous for A 174, thus displaying His 27 (Figures 11 a- l id, arrows), and the remaining group was heterozygous, namely containing G and A at nucleotide 174, thus expressing two RFC species, one with Arg 27, and another with His 27 (Figures 1 la- 1 Id, arrowhead and arrows). Another DNA specimen obtained at diagnosis from a B-precursor

ALL patient (termed J7) displayed an altered electrophoretic mobility pattern for exon 6 (Figure 10c, lane a). Sequencing revealed that this patient contained a G to A mutation at nucleotide 1635, which resulted in a substitution of Asn 522 for Asp in the C-terminus region of RFC (Table 4, above). Comparison of MTX uptake in blast cells from B-precursor ALL patient J7 and other leukemia patients having intact RFC (Figure lOd) clearly demonstrates impaired antifolate transport resulting from the Asp522Asn mutation. As would be expected in view of the findings described herein, patient J7 was fatally chemotherapy resistant. Transfection of aberrant RFC cDNA into MTX-A cells:

To study the role of the His 56 mutation identified in the ALL patient, as well as the polymorphic His 27 variation, on antifolate resistance and folate growth requirement, RFC cDNAs harboring either or both of these genetic alterations, were stably transfected into transport-deficient mouse leukemia MTX^rA cells. A dominant mutant was used as control: the Glu 45 Lys RFC mutation recently shown to play a major contributing role in the >200-fold MTX resistance, due to a severe impairment of MTX transport while preserving folic acid and leucovorin uptake (Jansen et al, 1998). Thus, a parameter was defined, calculated as the ratio of MTX growth inhibition EC₅₀ by the folate