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Combination of JAK2 and HSP90 inhibitors: an effective therapeutic option in drug-resistant chronic myelogenous leukemia

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ABSTRACT

Recent studies suggest that JAK2 serves as a novel therapeutic target in Bcr-Abl+ chronic myelogenous leukemia (CML). We have reported the existence of an HSP90- associated high molecular weight network complex (HMWNC) that is composed of HSP90 client proteins BCR-ABL, JAK2, and STAT3 in wild type Bcr-Abl+ leukemic cells. Here we showed that the HSP90-HMWNC is present in leukemia cells from CML patients in blast stage, and in Imatinib (IM)-resistant 32Dp210 (T315I) leukemia cells. We found that the HSP90-HMWNC could be disassembled by depleting JAK2 with either Jak2-specific shRNA or treatment with JAK2 inhibitors (TG101209 or Ruxolitinib) and HSP90 inhibitor (AUY922). Combinational treatment with JAK2 and HSP90 inhibitors diminished the activation of BCR-ABL, JAK2 and its downstream targets. As a result, the IM-resistant 32Dp210 T315I cells underwent apoptosis. When administered in mice bearing 32Dp210 T315I leukemia, combinational therapy using Ruxolitinib and AUY922 prolonged the survival significantly. Thus, a combination of JAK2 and HSP90 inhibitors could be a powerful strategy for the treatment of CML, especially in IM-resistant patients.

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Combinational treatment using both HSP90 and JAK2 inhibitors enhanced the inhibition of the BCR- ABL signaling in IM-resistant 32Dp210 T315I cells(A-D) IM-resistant 32Dp210 T315I cells were treated with JAK2 inhibitor TG101209 (100nM) with different dosages of HSP90 inhibitor AUY922 (0-40nM) for 6hr. Western blotting was performed using antibodies against pBCR-ABL (4G10) and pJAK2 (Tyr1007/1008) (A); pLYN (B); BCR-ABL pTyr177 (C); and pAKT (D). (E) Decreased levels of BCR-ABL and JAK2 in HSP90-immunoprecipitation complex from 32Dp210 T315I cells treated with JAK2 inhibitor or HSP90 inhibitor, or both; (E-F) A different JAK inhibitor Ruxolitinib (10μM) showed synergistic effect with HSP90 inhibitor AUY922 (20nM) in blocking JAK2 activity as measured by pJAK2 (Tyr1007/1008) (E), BCR-ABL activity as measured by pTyr177, and LYN activity as measured by pTyr396 (F).
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Figure 2: Combinational treatment using both HSP90 and JAK2 inhibitors enhanced the inhibition of the BCR- ABL signaling in IM-resistant 32Dp210 T315I cells(A-D) IM-resistant 32Dp210 T315I cells were treated with JAK2 inhibitor TG101209 (100nM) with different dosages of HSP90 inhibitor AUY922 (0-40nM) for 6hr. Western blotting was performed using antibodies against pBCR-ABL (4G10) and pJAK2 (Tyr1007/1008) (A); pLYN (B); BCR-ABL pTyr177 (C); and pAKT (D). (E) Decreased levels of BCR-ABL and JAK2 in HSP90-immunoprecipitation complex from 32Dp210 T315I cells treated with JAK2 inhibitor or HSP90 inhibitor, or both; (E-F) A different JAK inhibitor Ruxolitinib (10μM) showed synergistic effect with HSP90 inhibitor AUY922 (20nM) in blocking JAK2 activity as measured by pJAK2 (Tyr1007/1008) (E), BCR-ABL activity as measured by pTyr177, and LYN activity as measured by pTyr396 (F).

Mentions: We have showed that the HSP90-HMWNC is sensitive to HSP90 inhibitor AYU922, which leads to the disassembly of the multiprotein complex and cell death in 32Dp210 cells [24]. When treating IM-resistance 32Dp210 T315I cells with either JAK2 inhibitor TG101209 (100nM) or HSP90 inhibitor AUY922 (20nM) alone, the association of JAK2 with HSP90 was decreased (Figure 2D). The loss of JAK2 and BCR-ABL in HSP90 immunoprecipitation (IP) complex was more apparent in cells treated with both drugs (Figure 1D, lane 4). Furthermore, this combinational treatment led to the disassembly of the HSP90-HMWNC, as evidenced by the analysis using gel filtration chromatography (Figure 1F). In cells treated with both drugs for a shorter time (6hr), HSP90-HMWNC was relatively intact (Figure 1E). When the cells were treated for longer hours (16hr), the components of the HMWNC shifted towards a lower molecular weight region (Figure 1F). Interestingly, HSP90 was found to associate with some members of the complex such as STAT3 which was co-eluted in the same fractions (Figure 1F).


Combination of JAK2 and HSP90 inhibitors: an effective therapeutic option in drug-resistant chronic myelogenous leukemia
Combinational treatment using both HSP90 and JAK2 inhibitors enhanced the inhibition of the BCR- ABL signaling in IM-resistant 32Dp210 T315I cells(A-D) IM-resistant 32Dp210 T315I cells were treated with JAK2 inhibitor TG101209 (100nM) with different dosages of HSP90 inhibitor AUY922 (0-40nM) for 6hr. Western blotting was performed using antibodies against pBCR-ABL (4G10) and pJAK2 (Tyr1007/1008) (A); pLYN (B); BCR-ABL pTyr177 (C); and pAKT (D). (E) Decreased levels of BCR-ABL and JAK2 in HSP90-immunoprecipitation complex from 32Dp210 T315I cells treated with JAK2 inhibitor or HSP90 inhibitor, or both; (E-F) A different JAK inhibitor Ruxolitinib (10μM) showed synergistic effect with HSP90 inhibitor AUY922 (20nM) in blocking JAK2 activity as measured by pJAK2 (Tyr1007/1008) (E), BCR-ABL activity as measured by pTyr177, and LYN activity as measured by pTyr396 (F).
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Figure 2: Combinational treatment using both HSP90 and JAK2 inhibitors enhanced the inhibition of the BCR- ABL signaling in IM-resistant 32Dp210 T315I cells(A-D) IM-resistant 32Dp210 T315I cells were treated with JAK2 inhibitor TG101209 (100nM) with different dosages of HSP90 inhibitor AUY922 (0-40nM) for 6hr. Western blotting was performed using antibodies against pBCR-ABL (4G10) and pJAK2 (Tyr1007/1008) (A); pLYN (B); BCR-ABL pTyr177 (C); and pAKT (D). (E) Decreased levels of BCR-ABL and JAK2 in HSP90-immunoprecipitation complex from 32Dp210 T315I cells treated with JAK2 inhibitor or HSP90 inhibitor, or both; (E-F) A different JAK inhibitor Ruxolitinib (10μM) showed synergistic effect with HSP90 inhibitor AUY922 (20nM) in blocking JAK2 activity as measured by pJAK2 (Tyr1007/1008) (E), BCR-ABL activity as measured by pTyr177, and LYN activity as measured by pTyr396 (F).
Mentions: We have showed that the HSP90-HMWNC is sensitive to HSP90 inhibitor AYU922, which leads to the disassembly of the multiprotein complex and cell death in 32Dp210 cells [24]. When treating IM-resistance 32Dp210 T315I cells with either JAK2 inhibitor TG101209 (100nM) or HSP90 inhibitor AUY922 (20nM) alone, the association of JAK2 with HSP90 was decreased (Figure 2D). The loss of JAK2 and BCR-ABL in HSP90 immunoprecipitation (IP) complex was more apparent in cells treated with both drugs (Figure 1D, lane 4). Furthermore, this combinational treatment led to the disassembly of the HSP90-HMWNC, as evidenced by the analysis using gel filtration chromatography (Figure 1F). In cells treated with both drugs for a shorter time (6hr), HSP90-HMWNC was relatively intact (Figure 1E). When the cells were treated for longer hours (16hr), the components of the HMWNC shifted towards a lower molecular weight region (Figure 1F). Interestingly, HSP90 was found to associate with some members of the complex such as STAT3 which was co-eluted in the same fractions (Figure 1F).

View Article: PubMed Central - PubMed

ABSTRACT

Recent studies suggest that JAK2 serves as a novel therapeutic target in Bcr-Abl+ chronic myelogenous leukemia (CML). We have reported the existence of an HSP90- associated high molecular weight network complex (HMWNC) that is composed of HSP90 client proteins BCR-ABL, JAK2, and STAT3 in wild type Bcr-Abl+ leukemic cells. Here we showed that the HSP90-HMWNC is present in leukemia cells from CML patients in blast stage, and in Imatinib (IM)-resistant 32Dp210 (T315I) leukemia cells. We found that the HSP90-HMWNC could be disassembled by depleting JAK2 with either Jak2-specific shRNA or treatment with JAK2 inhibitors (TG101209 or Ruxolitinib) and HSP90 inhibitor (AUY922). Combinational treatment with JAK2 and HSP90 inhibitors diminished the activation of BCR-ABL, JAK2 and its downstream targets. As a result, the IM-resistant 32Dp210 T315I cells underwent apoptosis. When administered in mice bearing 32Dp210 T315I leukemia, combinational therapy using Ruxolitinib and AUY922 prolonged the survival significantly. Thus, a combination of JAK2 and HSP90 inhibitors could be a powerful strategy for the treatment of CML, especially in IM-resistant patients.

No MeSH data available.


Related in: MedlinePlus