RNAi screening of the kinome with cytarabine in leukemias

Abstract

To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

Figure 1

Experimental overview of siRNA screens.

Figure 2

Performance and robustness of RNAi screens. (A) Reproducibility with Pearson correlation coefficient of R = 0.65 for duplicate RNAi screens conducted in THP-1 at target EC₃₀. (B) Transfection efficiency for TF-1 and THP-1. Reduction in relative cell number was calculated as a percentage of the universal lethal control (n = 16) versus the median value for all 572 siRNA as a readout for transfection efficiency. (C) Ara-C growth-inhibitory effects. Viability calculated as the percentage inhibition of the median of NS-siRNA (n = 16) over NS-siRNA + Ara-C (n = 16). (D) Nonspecific effects on relative cell number calculated as the fraction of NS-siRNA (median, n = 16) versus buffer/control medium for both NS-siRNA–only and NS-siRNA + Ara-C. TF-1 screens were performed at higher (EC₇₀) and lower (EC₃₀) Ara-C concentrations. RLUs indicates relative luminescene units.

Figure 3

siRNA screen hits. Venn diagram of hits from primary siRNA-kinome sensitizer screens with Ara-C. Genes/hits depicted in bold are common Ara-C sensitizers identified in both TF-1 and THP-1.

Figure 4

Validation of hits by secondary siRNA screens. Validation of selected top hits from primary siRNA screens with 4 siRNA against each gene. Reduction in relative cell number expressed as Ara-C with siRNA versus Ara-C alone. NS indicates the nonsilencing control. Darkly shaded bars correspond to TF-1 and lightly shaded bars to THP-1.

Figure 5

Target silencing by siRNA. (A) WEE1 and CHEK1 transcript levels after treatment with siRNA targeting WEE1 or CHEK1 compared with NS-siRNA. (B) Immunoblots of extracts from cells treated with WEE1 or CHEK1 siRNA with and without 270nM Ara-C. WEE1 (top panel) and CHEK1 (bottom panel). NS indicates the nonsilencing control; and UT, untreated control.

Figure 6

MK1775 is synergistic with Ara-C ex vivo. Percentage inhibition of relative cell number for the combination of Ara-C and MK1775 over Ara-C alone in primary AML, MDS, and CML patient samples. (A) Initial 4 (of 5) primary samples as described in the text. (B) AML and MDS primary samples. (C) CML primary samples. For panels B and C, the maximum percentage inhibition is shown for any concentration of MK1775 or Ara-C (darkly shaded bars) and for the greatest inhibition observed at or below 380nM MK1775 with any concentration of Ara-C (lightly shaded bars; see “MK1775 sensitizes primary AML, MDS, and CML specimens to Ara-C ex vivo” details).

Figure 7

Ara-C and MK1775 induce apoptosis. Percentage of cleaved caspase-3 (CC3)-positive cells after Ara-C, MK1775, or combination treatment in THP-1 cells (A), MDS-L cells (B), and a primary AML sample (C).