Cytokine receptor signaling is required for the survival of ALK- anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations

Abstract

Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)^- anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK- ALCL, we treated ALK- cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK- ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK- ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK- ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK- ALCL who are phosphorylated STAT3<sup/>.

Keywords: ALK− ALCL; JAK; STAT; cytokine receptor; mutations.

Fig. 1.

JAK inhibitors diminished various p-STAT3⁺ ALK− ALCL cell line growth in vitro. (A) JAK inhibitors ruxolitinib, tofacitinib, and AZ-3 inhibited cell proliferation in most exogenous cytokine-independent ALK− ALCL cells. The cells were treated with a series of increasing concentrations of inhibitors. Blue, ALK+ ALCL cell lines; red, ALK− ALCL cell lines. The data are representative of three independent experiments. (B) Western blot analysis of STAT3 phosphorylation in ALK− ALCL cells. (C) Tofacitinib treatment decreased STAT3 phosphorylation in ALK− ALCL cells. The cells were treated with tofacitinib (1 μg/mL) for 4 h before harvesting.

Fig. 2.

JAK1 (and JAK2) are required for cell survival in JAK inhibitor-sensitive cells. (A) shJAK1 induced cell death in all JAK inhibitor-sensitive cells. The percentage of viable shRNA+ cells at days 2, 4, 6, 8, 10, and 12 after addition of doxycycline was compared with that of day 0. shRNA-infected cells were GFP⁺; the shRNA expression was induced by doxycycline. shSC4 served as a negative control; shRPL6, as a positive control. (B) shJAK2 induced cell death in Mac-1/2A/2B cells. (C) Western blot analysis of JAK1 and p-STAT3 in Mac-1 cells stably infected with retroviral shJAK1. The shRNA expression was induced by doxycycline for 24 h or 72 h. (D) Western blot analysis of PCM1-JAK2 and p-STAT3 in Mac-1 cells stably infected with retroviral shJAK2.

Fig. 3.

STAT3 is required for cell survival in all JAK inhibitor-sensitive cells. (A) shSTAT3 induced cell death in all JAK inhibitor-sensitive cells. (B) Western blot analysis of STAT3 expression and p-STAT3 in Mac-1 cells stably infected with shSTAT3.

Fig. S1.

Western blot analysis of JAK2 and PCM1-JAK2.

Fig. 4.

JAK1 and STAT3 mutations induce increased STAT3 activity in response to IL-6. (A) JAK1G1097V induced increased STAT3 reporter activity compared with WT JAK1 in response to IL-6 (JAK1 vs. JAK1G1097V, P = 0.02). (B) JAK1G1097V induced greater STAT3 phosphorylation in response to IL-6. (C) STAT3 mutants induced increased STAT3 reporter activity in response to IL-6 compared with WT STAT3 (STAT3 vs. G618R, P < 0.01; STAT3 vs. S614R, P = 0.03; STAT3 vs. D661Y, P < 0.01). (D) STAT3 mutants induced increased STAT3 phosphorylation in response to IL-6. IL-6 (10 ng/mL) was added (or not) to the cell culture at 30 min before harvesting.

Fig. 5.

WT JAK1 or STAT3 is capable of promoting cell growth in JAK1/STAT3 mutant-containing FE-PD cells. (A) WT JAK1 abrogated cell death induced by shJAK1. FE-PD cells that stably expressed WT were infected with shJAK1 to knock down endogenous JAK1 expression. Cell growth was monitored in infected cells (GFP⁺). shJAK1 expression was induced by doxycycline. (B) Western blot analysis of JAK1 expression in WT and JAK1-overexpressing cells. (C) WT STAT3 abrogated cell death induced by shSTAT3. (D) Western blot analysis of STAT3 expression in WT and STAT3-overexpressing cells.

Fig. S2.

Replacing endogenous JAK1 with WT JAK 1 did not affect pSTAT3 Y705 or pSTAT3 S727 expression. **Endogenous JAK1 was knocked down by shJAK1, whereas WT JAK1 was not knocked down.

Fig. S3.

WT STAT3 abrogated cell death induced by shSTAT3 in TLBR2 cells.

Fig. S4.

IL-6 and γ cytokine receptor expression in JAK inhibitor-sensitive ALK− ALCL cells.

Fig. 6.

Cytokine receptor GP130 and IL-2Rγ are needed for cell survival in select JAK inhibitor-sensitive cells. (A) shGP130 induced cell death in Mac-1/2A/2B cells. (B) shGP130 induced decreased STAT3 phosphorylation in Mac-1/2A/2B cells. (C) shGP130 depleted surface expression of GP130. The black dotted line represents IgG control; the black solid line, before shGP130 induction; red solid line, 72 h after shGP130 induction. (D) shIL-2Rγ induced cell death in FE-PD cells. (E) shIL-2Rγ induced decreased STAT3 phosphorylation in FE-PD cells. (F) shIL-2Rγ depleted surface expression of IL-2Rγ. The black dotted line represents IgG control; the black solid line, before shGP130 induction; the red solid line, 72 h after shGP130 induction.

Fig. S5.

Cytokine gene expression is increased in primary ALK− ALCL tumors. Cytokine gene expression was measured by Nanostring. The expression levels were normalized to levels in 15 housekeeping genes and were compared with those of normal T cells.

Fig. 7.

Ruxolitinib inhibits tumor growth in a xenograft ALK− ALCL mouse model. (A) Ruxolitinib inhibited ALK− ALCL tumor growth in vivo. Ruxolitinib was given at 50 mg/kg/d continuously via a pump (Alzet) for 7 d. Treatment started when the tumor volume reached 100 mm³. Control vs. ruxolitinib, P < 0.01. (B) Ruxolitinib treatment decreased tumor weight when measured at day 9. Control vs. ruxolitinib, P < 0.001.

Fig. S6.

Neutralizing antibodies to the γc cytokine/receptor, IL-6 family cytokine/receptor does not affect cell growth in JAK inhibitor-sensitive ALK− ALCL cells.