STROMA-DERIVED FACTORS STIMULATE JAK/STAT SIGNALING IN AML CELLS RESULTING IN RESISTANCE TO BCL2 INHIBITOR VENETOCLAX
Author(s): ,
Riikka Karjalainen
Affiliations:
Institute for Molecular Medicine Finland, FIMM,Helsinki,Finland
,
Mihaela Popa
Affiliations:
Centre for Cancer Biomarkers CCBIO, Department of Clinical Science,University of Bergen,Bergen,Norway
,
Minxia Liu
Affiliations:
Institute for Molecular Medicine Finland, FIMM,Helsinki,Finland
,
Komal Kumar Javarappa
Affiliations:
Institute for Molecular Medicine Finland, FIMM,Helsinki,Finland
,
Mika Kontro
Affiliations:
Hematology Research Unit Helsinki,University of Helsinki,Helsinki,Finland;Department of Hematology,Helsinki University Hospital Comprehensive Cancer Center,Helsinki,Finland
,
Alun Parsons
Affiliations:
Institute for Molecular Medicine Finland, FIMM,Helsinki,Finland
,
Kimmo Porkka
Affiliations:
Hematology Research Unit Helsinki,University of Helsinki,Helsinki,Finland;Department of Hematology,Helsinki University Hospital Comprehensive Cancer Center,Helsinki,Finland
,
Krister Wennerberg
Affiliations:
Institute for Molecular Medicine Finland, FIMM,Helsinki,Finland
,
Emmet McCormack
Affiliations:
Centre for Cancer Biomarkers CCBIO, Department of Clinical Science,University of Bergen,Bergen,Norway;Department of Internal Medicine,Haukeland University Hospital,Bergen,Norway
,
Bjørn Tore Gjertsen
Affiliations:
Centre for Cancer Biomarkers CCBIO, Department of Clinical Science,University of Bergen,Bergen,Norway
Caroline A. Heckman
Affiliations:
Institute for Molecular Medicine Finland, FIMM,Helsinki,Finland
(Abstract release date: 05/18/17) EHA Library. Karjalainen R. 06/23/17; 181468; P181
Riikka Karjalainen
Riikka Karjalainen
Contributions
Abstract

Abstract: P181

Type: Poster Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 17:15 - 18:45

Location: Poster area (Hall 7)

Background
The bone marrow (BM) microenvironment is known to protect AML cells from drug therapy. We showed earlier that conditioned medium (CM) from the BM stromal cell line HS-5 increased cell viability and led to resistance to specific drug classes.

Aims

Here, we investigate the mechanisms mediating the BM stromal cell induced resistance to venetoclax and its reversal by ruxolitinib.
 

Methods
Phospho-flow analysis was done by stimulating AML patient cells with GM-CSF, G-CSF, IL-6, IL-8 or MIP-3α (10 ng/mL) for 20 min, after which the cells were stained with Alexa 647-anti-phospho-Stat5 (pY694), PE188 CF594-anti-phospho-Stat3 (pY705), BV421-anti-phospho-Akt (pS473) and PE-anti-phospho-Erk1/2 (pT202/pY204). For co-culture and transwell assays AML cells were added directly to MSCs from AML patients or separated by a 0.4 μm pore membrane. Vehicle (DMSO), ruxolitinib (300 nM), venetoclax (100 nM) or their combination were incubated for 48h and AML cells labeled with PE-Annexin V, 7AAD, PE-Cy7-CD34, BV605-CD45. In vivo drug efficacy was tested on NSG mice inoculated i.v. with MOLM-13luc AML cells. Mice were divided into control, venetoclax (25 mg/kg, i.p.), ruxolitinib (50 mg/kg BID, p.o) and combination groups (all n = 6) and treated for 3 weeks, 5 days a week with 2 days off.

Results

To identify the factors contributing to BM mediated drug resistance of AML cells, we analyzed the effect of IL-6, IL-8, MIP-3α, GM-CSF and G-CSF, cytokines enriched in the HS-5 CM, on proliferation of MNCs collected from AML patients. GM-CSF and to some extent G-CSF alone conferred resistance to venetoclax similar to CM that we showed earlier to reduce sensitivity to BCL2 inhibitors. To identify the impact of stroma-derived factors on cellular signaling we stimulated AML patient cells with CM and analyzed the phosphorylation of STAT3, STAT5, ERK and AKT. Compared to control conditions, CM rapidly induced phosphorylation of STAT5 in primary AML cells. When the effect of individual cytokines was tested, we noted that GM-CSF and G-CSF alone could mimic the effect of CM on cellular signaling. Gene expression data showed the receptor for GM-CSF (CSFR2A) is more highly expressed in AML patient cells compared to healthy controls. Taken together, these results show that cytokines such as GM-CSF from BM stromal cells increase JAK/STAT signaling, which may lead to enhanced survival of AML cells.
To determine whether the protective effect of stroma on BCL2 inhibition was dependent on cell-to-cell interactions we cultured AML patient cells either in direct contact with MSCs or separated from stroma with a 0.4 μm pore membrane. 48h treatment with 100 nM venetoclax did not result in significant reduction of CD34+ AML cells regardless of whether AML cells were directly cultured with stroma or separated by a membrane, further indicating that stroma-derived soluble factors are sufficient to reduce sensitivity to venetoclax.
Since the most abundant cytokines secreted by HS-5 cells, GM-CSF and G-CSF led to increased phosphorylation of STAT5, a downstream effector of JAKs, we tested a combination of venetoclax and JAK1/2 inhibitor ruxolitinib. We found that ruxolitinib potentiated sensitivity to venetoclax when tested with AML patient cells in HS-5 CM and in co-culture and transwell assays. Significantly, the combination was more effective at reducing tumor burden in a xenograft mouse model of AML than either drug alone.

Conclusion

In conclusion, our data demonstrate that BM secreted soluble factors drive cytoprotection against BCL2 antagonist venetoclax that can be overcome by combined blockade of JAK/STAT and BCL2 pathways with ruxolitinib and ventoclax in ex vivo co-culture models and in vivo in an AML mouse model. 

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): Drug resistance, Bone microenvironment, BCL2, Acute Myeloid Leukemia

Abstract: P181

Type: Poster Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 17:15 - 18:45

Location: Poster area (Hall 7)

Background
The bone marrow (BM) microenvironment is known to protect AML cells from drug therapy. We showed earlier that conditioned medium (CM) from the BM stromal cell line HS-5 increased cell viability and led to resistance to specific drug classes.

Aims

Here, we investigate the mechanisms mediating the BM stromal cell induced resistance to venetoclax and its reversal by ruxolitinib.
 

Methods
Phospho-flow analysis was done by stimulating AML patient cells with GM-CSF, G-CSF, IL-6, IL-8 or MIP-3α (10 ng/mL) for 20 min, after which the cells were stained with Alexa 647-anti-phospho-Stat5 (pY694), PE188 CF594-anti-phospho-Stat3 (pY705), BV421-anti-phospho-Akt (pS473) and PE-anti-phospho-Erk1/2 (pT202/pY204). For co-culture and transwell assays AML cells were added directly to MSCs from AML patients or separated by a 0.4 μm pore membrane. Vehicle (DMSO), ruxolitinib (300 nM), venetoclax (100 nM) or their combination were incubated for 48h and AML cells labeled with PE-Annexin V, 7AAD, PE-Cy7-CD34, BV605-CD45. In vivo drug efficacy was tested on NSG mice inoculated i.v. with MOLM-13luc AML cells. Mice were divided into control, venetoclax (25 mg/kg, i.p.), ruxolitinib (50 mg/kg BID, p.o) and combination groups (all n = 6) and treated for 3 weeks, 5 days a week with 2 days off.

Results

To identify the factors contributing to BM mediated drug resistance of AML cells, we analyzed the effect of IL-6, IL-8, MIP-3α, GM-CSF and G-CSF, cytokines enriched in the HS-5 CM, on proliferation of MNCs collected from AML patients. GM-CSF and to some extent G-CSF alone conferred resistance to venetoclax similar to CM that we showed earlier to reduce sensitivity to BCL2 inhibitors. To identify the impact of stroma-derived factors on cellular signaling we stimulated AML patient cells with CM and analyzed the phosphorylation of STAT3, STAT5, ERK and AKT. Compared to control conditions, CM rapidly induced phosphorylation of STAT5 in primary AML cells. When the effect of individual cytokines was tested, we noted that GM-CSF and G-CSF alone could mimic the effect of CM on cellular signaling. Gene expression data showed the receptor for GM-CSF (CSFR2A) is more highly expressed in AML patient cells compared to healthy controls. Taken together, these results show that cytokines such as GM-CSF from BM stromal cells increase JAK/STAT signaling, which may lead to enhanced survival of AML cells.
To determine whether the protective effect of stroma on BCL2 inhibition was dependent on cell-to-cell interactions we cultured AML patient cells either in direct contact with MSCs or separated from stroma with a 0.4 μm pore membrane. 48h treatment with 100 nM venetoclax did not result in significant reduction of CD34+ AML cells regardless of whether AML cells were directly cultured with stroma or separated by a membrane, further indicating that stroma-derived soluble factors are sufficient to reduce sensitivity to venetoclax.
Since the most abundant cytokines secreted by HS-5 cells, GM-CSF and G-CSF led to increased phosphorylation of STAT5, a downstream effector of JAKs, we tested a combination of venetoclax and JAK1/2 inhibitor ruxolitinib. We found that ruxolitinib potentiated sensitivity to venetoclax when tested with AML patient cells in HS-5 CM and in co-culture and transwell assays. Significantly, the combination was more effective at reducing tumor burden in a xenograft mouse model of AML than either drug alone.

Conclusion

In conclusion, our data demonstrate that BM secreted soluble factors drive cytoprotection against BCL2 antagonist venetoclax that can be overcome by combined blockade of JAK/STAT and BCL2 pathways with ruxolitinib and ventoclax in ex vivo co-culture models and in vivo in an AML mouse model. 

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): Drug resistance, Bone microenvironment, BCL2, Acute Myeloid Leukemia

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