Characterization of a mantle cell lymphoma cell line resistant to the Chk1 inhibitor PF-00477736

Abstract

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the chromosomal translocation t(11;14) that leads to constitutive expression of cyclin D1, a master regulator of the G1-S phase. Chk1 inhibitors have been recently shown to be strongly effective as single agents in MCL. To investigate molecular mechanisms at the basis of Chk1 inhibitor activity, a MCL cell line resistant to the Chk1 inhibitor PF-00477736 (JEKO-1 R) was obtained and characterized. The JEKO-1 R cell line was cross resistant to another Chk1 inhibitor (AZD-7762) and to the Wee1 inhibitor MK-1775. It displayed a shorter doubling time than parental cell line, likely due to a faster S phase. Cyclin D1 expression levels were decreased in resistant cell line and its re-overexpression partially re-established PF-00477736 sensitivity. Gene expression profiling showed an enrichment in gene sets involved in pro-survival pathways in JEKO-1 R. Dasatinib treatment partly restored PF-00477736 sensitivity in resistant cells suggesting that the pharmacological interference of pro-survival pathways can overcome the resistance to Chk1 inhibitors. These data further corroborate the involvement of the t(11;14) in cellular sensitivity to Chk1 inhibitors, fostering the clinical testing of Chk1 inhibitors as single agents in MCL.

Keywords: Chk1; cyclin D1; mantle cell lymphoma; mechanisms of resistance; targeted therapy.

Figure 1. Pharmacological activity of JEKO-1 cell line resistant to PF-00477736

Cytotoxic effect of PF-00477736 (A), AZD-7762 (B), MK-1776 (C), Bendamustine (D) and Bortezomib (E) in JEKO-1 parental (□) and in JEKO-1 R (■). Data are represented as mean ± SD of three independent experiments.

Figure 2. Analysis of cell cycle distribution

A. Cell growth curves of JEKO-1 parental (◊) and JEKO-1 R (■). Data are represented as mean ± SD of two independent experiments. B. Flow cytometric analysis of DNA content at 24, 48 and 72 hrs after seeding. Percentage of cell cycle phases (G1-S-G2/M) are included in the figure. As cells were in exponential growth, DNA distribution remained almost constant over time. The arrow points early S phase in JEKO-1 parental and the more evident G2-M peak in the JEKO-1 R cell line. C. Pulse-chase DNA-BrdUrd analysis at the end of 20 min of BrdUrd incubation (0 hr) and 7 hr after BrdUrd washout. Cells were considered BrdUrd-positive (BrdUrd+) when detected above the line. The vertical dashed line separates undivided (right) from divided (left) BrdUrd+ cells. The percentage of labeled undivided cells (%LU) and the relative movement (RM) are indicated in the plots. %LU represents those cells that were in S phase at 0 hr and that are still undivided after 7 hr, while RM represents their average state of completion of DNA duplication. D. Table summarizing the doubling time and duration of the different cell cycle phases in JEKO-1 parental and resistant cell lines.

Figure 3. Expression of cell cycle markers

A. Real time PCR showing cyclinA, Cdt1 and cyclin D1 expression levels in parental JEKO-1 cells (white bar) and in JEKO-1 R cells (grey bar). Data are normalized to the internal mRNA levels of actin and are represented as the fold change from JEKO-1 parental samples. Mean ±SD of three independent experiments. B. Western Blot Analysis showing cyclin A, Cdt1 and cyclin D1 and actin protein levels in the parental and resistant cell line. C. Western Blot Analysis showing pS317Chk1, Chk1 and actin protein levels in the JEKO-1 and JEKO-1 R cell lines.

Figure 4. Modulation of Cyclin D1 expression in the resistant JEKO-1 cell line

A. Real time PCR (upper panel) and western Blot Analysis (lower panel) of cyclin D1 levels in parental JEKO-1 (white bar), JEKO-1 R non infected (black bar) and infected with either control (light grey bar) or cyclin D1 lentiviral vector (grey bar). B. Cytotoxic effect of PF-00477736 in parental JEKO-1, JEKO-1 R not infected and infected with either control or cyclinD expressing lentiviral vector. Data are represented as mean ± SD of two independent experiments.

Figure 5. SRC pathway in the JEKO-1 R cell line

A. Real Time PCR of FGR levels in JEKO-1 parental and resistant cells. Data are represented as mean ± SD. B. Cytotoxic effect of PF-00477736 in parental (left panel) and resistant (right panel) JEKO-1 cells either alone or with not toxic concentration of Dasatinib. Data are represented as mean ± SD.

Figure 6. Model of Chk1 role in JEKO-1 parental and resistant cell line

A. JEKO-1 parental cells with the chromosomal translocation t(11;14), have an enhanced G1-S transition due to cyclinD1 constitutive expression and are strongly dependent on Chk1 kinase which plays a crucial role in control of initiation of DNA replication and in the regulation of correct progression into S phase, minimizing endogenous DNA damage. Thus Chk1 inhibition in JEKO-1 cell line leads to cell death B.. JEKO-1 R cells showed a decrease in expression of cyclin D1 which correlates with a lower rate of G1-S transition, thus they became less dependent on Chk1 activation for a correct initiation of DNA replication and progression into S phase which proceeds quicker in the resistant cell line. Chk1 is not constitutively activated in JEKO-1 R cells and its inhibition is not lethal (see text).