Pharmacodynamics of cytarabine alone and in combination with 7-hydroxystaurosporine (UCN-01) in AML blasts in vitro and during a clinical trial

Abstract

Chk1 and Akt signaling facilitate survival of cells treated with nucleoside analogues. Activation of Chk1 in response to cytarabine (ara-C) induced an S-phase checkpoint characterized by the inhibition of Cdk2, cell cycle arrest, no change in constitutively active Akt, or low-stress kinase signaling in ML-1 cells. However, inhibition of Chk1 by UCN-01 in S-phase-arrested cells resulted in an abrogation of the checkpoint, inhibition of Akt, activation of JNK, and a rapid induction of apoptosis. Similarly, primary acute myelogenous leukemia (AML) blasts exposed to ara-C and UCN-01 demonstrated a selective loss in cloning potential when compared with normal progenitors. Therefore, we evaluated a pilot clinical trial of ara-C in combination with UCN-01 in patients with relapsed AML. Blasts from some patients demonstrated a previously activated Chk1-Cdk2 DNA damage response pathway that decreased during therapy. Constitutively phosphorylated Akt kinase declined on addition of UCN-01 to the ara-C infusion, an action accompanied by an activation of JNK and reduction in absolute AML blast counts. Thus, use of UCN-01 in combination with ara-C decreases Chk1 phosphorylation, inhibits the Akt survival pathway, and activates JNK during the course of therapy, offering a rationale for the cytotoxic action of this combination during AML treatment.

Figure 1.

S-phase arrest by ara-C: abrogation by UCN-01. Exponentially growing ML-1 cells were incubated with 50 nM ara-C for 24 hours (○), at which time the culture was split and UCN-01 (50 nM) was added to one portion (•). Cells were sampled from each culture at the indicated times. DNA content (A, S phase; B, G₂/M; C, G₁) and TUNEL reactivity (D) were analyzed by flow cytometry. Symbols represent mean ± SD.

Figure 2.

Activation of an S-phase checkpoint in response to ara-C: dysregulation by UCN-01 in ML-1 cells. Exponentially growing ML-1 cells were treated as in Figure 1. (A) pSer³¹⁷Chk1 (top), pSer³⁴⁵Chk1 (middle), and total Chk1 (bottom) in response to ara-C and UCN-01; (B) pTyr¹⁵Cdk2 (top) and total Cdk2 (bottom) in cells depleted for Cdk1 protein. (C) pSer⁴⁷³Akt (top), pSer³⁰⁸Akt (middle), and total Akt (bottom) and (D) p42/44^Erkkinase activity and (E) c-jun kinase activation as measured by endogenous c-jun phosphorylation (top) normalized to the levels of total c-jun (bottom).

Figure 3.

Clonogenic assays. (A) Effect of ara-C (⋄, ×) and ara-C in combination with UCN-01 (♦, ^*) on the clonogenic survival of CFU-GM progenitor cells from normal bone marrow. (B) Effect of ara-C alone (○, ▵, □, ▿), UCN-01 alone (^*, +, ×, |), and ara-C in combination with UCN-01 (•, ▴, ▪, ▾) on the clonogenic survival of AML blasts. Symbols indicate the mean of 2 separate determinations from individual patients.

Figure 4.

Pharmacodynamics of ara-C in combination with UCN-01. (A) Cellular accumulation of ara-CTP during therapy. (B) Inhibition of DNA synthesis during therapy. (C) Relationship between cellular concentrations of ara-CTP in circulating leukemic blasts 4 hours after ara-C infusion and extent of DNA synthesis inhibition relative to pretreatment values. ▴ indicates patient 1; ▪, patient 2; ▾, patient 3; ♦, patient 4; •, patient 5; □, patient 6; ⋄, patient 7; ○, patient 8;▵, patient 9; ^*, patient 10; ¦, patient 11; +, patient 12; and ×, patient 13.

Figure 5.

Response of the checkpoint, survival, and stress-activated kinases within a cell sample from patient 4. Effect of ara-C and UCN-01 on levels of pSer³⁴⁵Chk1, total Chk1, pTyr¹⁵Cdk2, total Cdk2, pSer⁴⁷³Akt, total Akt, and JNK activation from AML blasts of patient 4 during therapy.

Figure 6.

Effect of ara-C alone and in combination with UCN-01 on cell cycle checkpoint pathways. (A) Levels of pSer³⁴⁵Chk1 relative to total Chk1 and (B) pTyr¹⁵Cdk2 relative to total Cdk2 in samples from 8 individuals during therapy. See Figure 4 for explanation of symbols.

Figure 7.

Effect of ara-C alone and in combination with UCN-01 on survival pathways and stress-activated kinases. (A) pSer⁴⁷³Akt relative to total Akt levels in 8 individuals during therapy. (B) Change in JNK activity in 8 individuals during therapy measured as described.See Figure 4 for explanation of symbols.

Figure 8.

Response of leukemia cells to ara-c and UCN-01. Action of ara-C alone (A) or in combination with UCN-01 (B) on the Chk1, Akt, and JNK pathways in leukemia cells.