Figure 2.
Figure 2. Analysis of sensitivity to PKC412 in primary AML blasts obtained from patient UPN1. (A) In vivo tyrosine phosphorylation status of FLT3. MNCs were isolated from patient UPN1 at the time of remission (day 148) and at the time of recurrence of PB blasts (days 288, 289, 290). Whole cell lysates were prepared and were analyzed by Western blotting. Constitutive tyrosine phosphorylation of FLT3 was detected at the time points of relapse, but not in remission, on day 148.As a control, 32D cells harboring FLT3-ITD treated with or without PKC412 (100 nM, 30 minutes at 37°C) were used. To control equal loading, the blot was stripped and reprobed using an antiactin antibody. (B) Ex vivo bioassay for FLT3 tyrosine kinase inhibition. This immunoblot was derived from MV4;11 cells harboring the FLT3-ITD mutation exposed to serum from patient UPN1 and to serum from a healthy donor (HD) spiked with and without PKC412 (100 nM, 30 minutes at 37°C). Serum was obtained from patient UPN1 at various time points while he was on therapy with PKC412. Whole cell lysates were analyzed by Western blotting using FLT3-specific antiphosphotyrosine antibodies. Inhibition of the constitutive tyrosine phosphorylation was detectable using patient serum derived from all time points and healthy donor serum spiked with PKC412. Densitometric analysis revealed more than 95% inhibition of tyrosine-phosphorylated FLT3 levels at day 288. (C) FLT3 tyrosine phosphorylation in primary AML blasts treated with and without PKC412 and AST487. MNCs isolated from peripheral blood were incubated in RPMI 1640 with 10% FCS and were treated with increasing doses of PKC412 and the novel FLT3-TKI AST487. Whole cell lysates were analyzed by Western blotting using FLT3-specific antiphosphotyrosine antibodies. (D) Analysis of induction of apoptosis in primaryAML blasts using PKC412 andAST487. MNCs obtained from patient UPN1 at the time relapse were maintained in RPMI 1640 without any growth factor supplementation and were incubated with or without PKC412 (50 nM), FL (100 ng/mL), AST487 (50 nM), or in combination for 90 hours, as indicated. After this, the percentage of sub-G1 cells corresponding to apoptotic cells was determined in triplicate by flow cytometry. Error bars correspond to standard deviation.

Analysis of sensitivity to PKC412 in primary AML blasts obtained from patient UPN1. (A) In vivo tyrosine phosphorylation status of FLT3. MNCs were isolated from patient UPN1 at the time of remission (day 148) and at the time of recurrence of PB blasts (days 288, 289, 290). Whole cell lysates were prepared and were analyzed by Western blotting. Constitutive tyrosine phosphorylation of FLT3 was detected at the time points of relapse, but not in remission, on day 148.As a control, 32D cells harboring FLT3-ITD treated with or without PKC412 (100 nM, 30 minutes at 37°C) were used. To control equal loading, the blot was stripped and reprobed using an antiactin antibody. (B) Ex vivo bioassay for FLT3 tyrosine kinase inhibition. This immunoblot was derived from MV4;11 cells harboring the FLT3-ITD mutation exposed to serum from patient UPN1 and to serum from a healthy donor (HD) spiked with and without PKC412 (100 nM, 30 minutes at 37°C). Serum was obtained from patient UPN1 at various time points while he was on therapy with PKC412. Whole cell lysates were analyzed by Western blotting using FLT3-specific antiphosphotyrosine antibodies. Inhibition of the constitutive tyrosine phosphorylation was detectable using patient serum derived from all time points and healthy donor serum spiked with PKC412. Densitometric analysis revealed more than 95% inhibition of tyrosine-phosphorylated FLT3 levels at day 288. (C) FLT3 tyrosine phosphorylation in primary AML blasts treated with and without PKC412 and AST487. MNCs isolated from peripheral blood were incubated in RPMI 1640 with 10% FCS and were treated with increasing doses of PKC412 and the novel FLT3-TKI AST487. Whole cell lysates were analyzed by Western blotting using FLT3-specific antiphosphotyrosine antibodies. (D) Analysis of induction of apoptosis in primaryAML blasts using PKC412 andAST487. MNCs obtained from patient UPN1 at the time relapse were maintained in RPMI 1640 without any growth factor supplementation and were incubated with or without PKC412 (50 nM), FL (100 ng/mL), AST487 (50 nM), or in combination for 90 hours, as indicated. After this, the percentage of sub-G1 cells corresponding to apoptotic cells was determined in triplicate by flow cytometry. Error bars correspond to standard deviation.

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