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. 2014 Jun;124(6):2762-73.
doi: 10.1172/JCI74182. Epub 2014 May 8.

Combined MEK and JAK inhibition abrogates murine myeloproliferative neoplasm

Guangyao Kong, Mark Wunderlich, David Yang, Erik A Ranheim, Ken H Young, Jinyong Wang, Yuan-I Chang, Juan Du, Yangang Liu, Sin Ruow Tey, Xinmin Zhang, Mark Juckett, Ryan Mattison, Alisa Damnernsawad, Jingfang Zhang, James C Mulloy, Jing Zhang

Combined MEK and JAK inhibition abrogates murine myeloproliferative neoplasm

Guangyao Kong et al. J Clin Invest. 2014 Jun.

Abstract

Overactive RAS signaling is prevalent in juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) in humans, and both are refractory to conventional chemotherapy. Conditional activation of a constitutively active oncogenic Nras (NrasG12D/G12D) in murine hematopoietic cells promotes an acute myeloproliferative neoplasm (MPN) that recapitulates many features of JMML and MP-CMML. We found that NrasG12D/G12D-expressing HSCs, which serve as JMML/MP-CMML-initiating cells, show strong hyperactivation of ERK1/2, promoting hyperproliferation and depletion of HSCs and expansion of downstream progenitors. Inhibition of the MEK pathway alone prolonged the presence of NrasG12D/G12D-expressing HSCs but failed to restore their proper function. Consequently, approximately 60% of NrasG12D/G12D mice treated with MEK inhibitor alone died within 20 weeks, and the remaining animals continued to display JMML/MP-CMML-like phenotypes. In contrast, combined inhibition of MEK and JAK/STAT signaling, which is commonly hyperactivated in human and mouse CMML, potently inhibited human and mouse CMML cell growth in vitro, rescued mutant NrasG12D/G12D-expressing HSC function in vivo, and promoted long-term survival without evident disease manifestation in NrasG12D/G12D animals. These results provide a strong rationale for further exploration of combined targeting of MEK/ERK and JAK/STAT in treating patients with JMML and MP-CMML.

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Figures

Figure 1
Figure 1. Endogenous NrasG12D/G12D induces hyperproliferation, decreased self-renewal, and depletion of HSCs.
(A–F) Control and NrasG12D/G12D (G/G) mice were treated with pI-pC and sacrificed at various time points (relative to the day of the first pI-pC injection, assigned as day 1) for analysis. Lin–CD41–CD48–cKit+Sca1+CD150+ HSCs (A), Lin–CD41–CD48–cKit+Sca1+CD150– MPPs (B), and LSK cells (C) were quantified using flow cytometry. SP, spleen; BM(H.L.), hind limb BM content (including tibias and femurs). WBM was estimated as 4-fold the hind limb BM value (42). (D and E) Cell cycle analysis of HSCs (D) and WBM (E) from control and NrasG12D/G12D mice using Ki67/DAPI staining on day 12. (F) A 16-hour pulse of EdU to quantify proliferating HSCs and WBM. (G) 20 HSCs purified from control or NrasG12D/G12D mice were transplanted with 2 × 105 congeneic BM cells into lethally irradiated mice. Donor-derived blood cells were regularly analyzed in the PB of recipients. (H) Donor-derived HSCs were quantified in recipients 12 weeks after transplantation. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2
Figure 2. NrasG12D/G12D hyperactivates ERK1/2 in HSCs, and downregulation of MEK/ERK signaling rescues NrasG12D/G12D HSC exhaustion.
Control and NrasG12D/G12D mice were treated with pI-pC and sacrificed on day 12. (A and B) CD150+CD41– cells (enriched for HSCs) and CD150–CD41– cells (enriched for MPPs) were sorted from Sca1+-enriched total BM cells. Sorted cells were serum and cytokine starved for 30 minutes at 37°C (A). IL-3 stimulation was performed for 10 minutes at 37°C after starvation (B). Levels of pERK1/2 and pAKT were measured using phosphospecific flow cytometry. HSCs (defined as [Lin CD48]–/locKit+ cells from sorted CD150+CD41– cells) and MPPs (defined as [Lin CD48]–/locKit+ cells from sorted CD150–CD41– cells) were gated for data analysis. To quantify the activation of ERK1/2, median intensities of pERK1/2 at different IL-3 concentrations are shown relative to the respective control cells at 0 ng/ml (assigned as 1). (C) Quantification of c-Myc and cell senescence–related genes in control and NrasG12D/G12D HSCs using qRT-PCR. (D) Quantification of BM spleen HSCs from control and NrasG12D/G12D mice treated with vehicle or AZD6244 for 7 days. (E) Single NrasG12D/G12D HSC genotyping after AZD6244 treatment. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
Figure 3. Combined inhibition of JAK and MEK effectively blocks the growth of human and mouse JMML/MP-CMML cells in vitro.
(A) WBM cells from moribund NrasG12D/G12D or KrasG12D/+ mice with advanced JMML/MP-CMML were serum and cytokine starved for 90 minutes at 37°C and incubated with DMSO vehicle or drugs for another 30 minutes at 37°C. Cells were then stimulated with 10 ng/ml GM-CSF or SCF for 10 minutes at 37°C. Levels of pERK1/2, pSTAT5, and pAKT were measured using phosphospecific flow cytometry. Non-neutrophil Lin–cKit+ cells were gated for data analysis. Shown are results of 1 representative experiment. (B and C) BM cells from moribund NrasG12D/G12D, NrasG12D/+, and KrasG12D/+ mice with advanced JMML/MP-CMML (B) and from human MP-CMML patients (C; n = 5) were cultured in triplicate in 96-well plates in the presence of vehicle or various concentrations of AZD6244 and/or AZD1480 for 5–14 days. Cell number was quantified by CellTiter-Glo assay. Data are mean ± SD.
Figure 4
Figure 4. Combined inhibition of JAK and MEK effectively controls the JMML/MP-CMML phenotypes in NrasG12D/G12D mice.
Control and NrasG12D/G12D mice were injected with pI-pC and then treated with vehicle, AZD6244, and/or AZD1480 from day 5 to day 12. (A) wbc number in PB and spleen weight. (B) Number of total HSCs and cycling HSCs (labeled with EdU-containing water [0.5 mg/ml] for the last 16 hours of drug treatment). (C) Number of CMPs and CLPs in BM and spleen. (D) Flow cytometric analysis of PB, spleen, and BM cells using myeloid lineage–specific markers. Debris and unlysed rbcs (low forward scatter) and dead cells (propidium iodide positive) were excluded from analysis. Significant differences from vehicle-treated controls (P < 0.05) are denoted by red font. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 5
Figure 5. Combined inhibition of JAK and MEK provides long-term survival in NrasG12D/G12D mice.
(A) Kaplan-Meier survival curves of NrasG12D/G12D mice with advanced JMML/MP-CMML treated with vehicle, AZD6244 alone, AZD1480 alone, or AZD6244 and AZD1480 combined; treatment was terminated either after 20 weeks or when mice reached a moribund stage. P values were determined by log-rank test. (B–F) Total wbc count (B), rbc count (C), hematocrit (D), hemoglobin level (E), and platelet count (F) in PB were measured every other week after treatment. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 6
Figure 6. Combined inhibition of JAK and MEK effectively controls JMML/MP-CMML phenotypes and prevents T-ALL development in NrasG12D/G12D mice.
NrasG12D/G12D mice with advanced JMML/MP-CMML were treated with vehicle, AZD6244 alone, AZD1480 alone, or AZD6244 and AZD1480 combined; treatment was terminated either after 20 weeks or when mice reached a moribund stage. (A–F) Total wbc count (A), spleen weight (B), thymus weight (C), HSC number (D), CMP number (E), and CLP number (F) in BM and spleen were quantified in different groups of animals. Because mice treated with low- or high-dose AZD6244 were essentially indistinguishable, we combined these 2 groups for data analysis. (G) Representative histologic H&E sections from spleen showed extensive infiltration of myelomonocytic cells and extramedullary hematopoiesis in NrasG12D/G12D mice treated with vehicle, AZD6244 alone, or AZD1480 alone, but not with AZD6244 and AZD1480 combined. Original magnification, ×4 (left); ×40 (right). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
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