Gabriele Stefanzl

Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34(+)/CD38(─) CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4(+) SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26(+) LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26(+) CML LSC engrafted NOD-SCID-IL-2Rγ(-/-) (NSG) mice with BCR/ABL1(+) cells, whereas CD26(─) SC from the same patients produced multilineage BCR/ABL1(-) engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1(+) cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy.

Identification of the Ki-1 antigen (CD30) as a novel therapeutic target in systemic mastocytosis

The Ki-1 antigen (CD30) is an established therapeutic target in patients with Hodgkin lymphoma and anaplastic large-cell lymphoma. We have recently shown that CD30 is expressed abundantly in the cytoplasm of neoplastic mast cells (MCs) in patients with advanced systemic mastocytosis (SM). In the current study, we asked whether CD30 is expressed on the surface of neoplastic MCs in advanced SM, and whether this surface structure may serve as therapeutic target in SM. As assessed by flow cytometry, CD30 was found to be expressed on the surface of neoplastic MCs in 3 of 25 patients (12%) with indolent SM, 4 of 7 patients (57%) with aggressive SM, and 4 of 7 patients (57%) with MC leukemia. The immature RAS-transformed human MC line MCPV-1.1 also expressed cell surface CD30, whereas the KIT-transformed MC line HMC-1.2 expressed no detectable CD30. The CD30-targeting antibody-conjugate brentuximab-vedotin inhibited proliferation in neoplastic MCs, with lower IC50 values obtained in CD30(+) MCPV-1.1 cells (10 µg/mL) compared with CD30(-) HMC-1.2 cells (>50 µg/mL). In addition, brentuximab-vedotin suppressed the engraftment of MCPV-1.1 cells in NSG mice. Moreover, brentuximab-vedotin produced apoptosis in all CD30(+) MC lines tested as well as in primary neoplastic MCs in patients with CD30(+) SM, but did not induce apoptosis in neoplastic MCs in patients with CD30(-) SM. Furthermore, brentuximab-vedotin was found to downregulate anti-IgE-induced histamine release in CD30(+) MCs. Finally, brentuximab-vedotin and the KIT D816V-targeting drug PKC412 produced synergistic growth-inhibitory effects in MCPV-1.1 cells. Together, CD30 is a promising new drug target for patients with CD30(+) advanced SM.

Target interaction profiling of midostaurin and its metabolites in neoplastic mast cells predicts distinct effects on activation and growth

Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multikinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC50 values. Midostaurin and CGP62221 also produced growth inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, which accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug competition experiments revealed that midostaurin interacts with KIT and several additional kinase targets. The key downstream regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE receptor downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation, which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs.

The pan‐Bcl‐2 blocker obatoclax promotes the expression of Puma, Noxa, and Bim mRNA and induces apoptosis in neoplastic mast cells

Advanced SM is an incurable neoplasm with short survival time. So far, no effective therapy is available for these patients. We and others have shown recently that neoplastic MC in ASM and MCL express antiapoptotic Mcl‐1, Bcl‐2, and Bcl‐xL. In this study, we examined the effects of the pan‐Bcl‐2 family blocker obatoclax (GX015‐070) on primary neoplastic MC, the human MC leukemia cell line HMC‐1, and the canine mastocytoma cell line C2. Obatoclax was found to inhibit proliferation in primary human neoplastic MC (IC50: 0.057 μM), in HMC‐1.2 cells expressing KIT D816V (IC50: 0.72 μM), and in HMC‐1.1 cells lacking KIT D816V (IC50: 0.09 μM), as well as in C2 cells (IC50: 0.74 μM). The growth‐inhibitory effects of obatoclax in HMC‐1 cells were accompanied by an increase in expression of Puma, Noxa, and Bim mRNA, as well as by apoptosis, as evidenced by microscopy, TUNEL assay, and caspase cleavage. Viral‐mediated overexpression of Mcl‐1, Bcl‐xL, or Bcl‐2 in HMC‐1 cells was found to introduce partial resistance against apoptosis‐inducing effects of obatoclax. We were also able to show that obatoclax synergizes with several other antineoplastic drugs, including dasatinib, midostaurin, and bortezomib, in producing apoptosis and/or growth arrest in neoplastic MC. Together, obatoclax exerts major growth‐inhibitory effects on neoplastic MC and potentiates the antineoplastic activity of other targeted drugs. Whether these drug effects can be translated to application in patients with advanced SM remains to be determined.

Identification of Ponatinib as a potent inhibitor of growth, migration, and activation of neoplastic eosinophils carrying FIP1L1-PDGFRA.

In chronic eosinophilic leukemia, the transforming oncoprotein FIP1L1-PDGFRA is a major target of therapy. In most patients, the tyrosine kinase inhibitor (TKI) imatinib induces complete remission. For patients who are intolerant or resistant, novel TKIs have been proposed. We examined the in vitro effects of 14 kinase blockers on growth and function of EOL-1 cells, a FIP1L1-PDGFRA(+) eosinophil cell line. Major growth-inhibitory effects were seen with all PDGFR-blocking agents, with IC50 values in the low nanomolar range: ponatinib, 0.1-0.2 nmol/L; sorafenib, 0.1-0.2 nmol/L; masitinib, 0.2-0.5 nmol/L; nilotinib, 0.2-1.0 nmol/L; dasatinib, 0.5-2.0 nmol/L; sunitinib, 1-2 nmol/L; midostaurin, 5-10 nmol/L. These drugs were also found to block activation of PDGFR-downstream signaling molecules, including Akt, S6, and STAT5 in EOL-1 cells. All effective TKIs produced apoptosis in EOL-1 cells as determined by microscopy, Annexin-V/PI, and caspase-3 staining. In addition, PDGFR-targeting TKIs were found to inhibit cytokine-induced migration of EOL-1 cells. In all bioassays used, ponatinib was found to be the most potent compound in EOL-1 cells. In addition, ponatinib was found to downregulate expression of the activation-linked surface antigen CD63 on EOL-1 cells and to suppress the growth of primary neoplastic eosinophils. We also examined drug effects on Ba/F3 cells expressing two clinically relevant, imatinib-resistant, mutant forms of FIP1L1-PDGFRA, namely T674I and D842V. Strong inhibitory effects on both mutants were seen only with ponatinib. In summary, novel PDGFR-targeting TKIs may be alternative agents for the treatment of patients with imatinib-resistant chronic eosinophilic leukemia. Although several different PDGFR-targeting agents are effective, the most potent drug appears to be ponatinib.

Identification of CD25 as STAT5-Dependent Growth Regulator of Leukemic Stem Cells in Ph+ CML.

Purpose: In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs. Experimental Design: CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25+ CML cell line KU812. Results: In contrast to normal hematopoietic stem cells, CD34+/CD38− CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin−/Sca-1+/Kit+ stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cells in vitro and their engraftment in vivo in NOD/SCID-IL-2Rγ−/− mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib. Conclusions: CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML. Clin Cancer Res; 22(8); 2051–61. ©2015 AACR.

BTK inhibition is a potent approach to block IgE-mediated histamine release in human basophils

Recent data suggest that Brutons tyrosine kinase (BTK) is an emerging therapeutic target in IgE receptor (IgER)‐cross‐linked basophils.

Maintenance therapy with histamine plus IL-2 induces a striking expansion of two CD56 bright NK cell subpopulations in patients with acute myeloid leukemia and supports their activation

Histamine dihydrochloride (HDC) plus IL-2 has been proposed as a novel maintenance-immunotherapy in acute myeloid leukemia (AML). We analyzed the immunophenotype and function of natural killer (NK) cells in blood of AML patients treated after chemotherapy with HDC plus IL-2. The treatment caused a striking expansion of CD56brightCD16neg and CD56brightCD16low NK cell subpopulations. A reduced NK cell fraction recovered and high proportions of cells expressed the activating receptors NKG2D, NKp30, and NKp46. Concomitantly, KIR-expressing NK cells were reduced and NK cells with inhibitory NKG2A/CD94 receptors increased beyond normal levels. In addition, the immunotherapy-induced NK cells exhibited high capacity to produce IFN-γ and to degranulate. Furthermore, we provide evidence from subsequent in vitro studies that this is caused in part by direct effects of IL-2 on the CD56bright cells. IL-2 specifically induced proliferation of both CD56bright subpopulations, but not of CD56dim cells. It further preserved the expression of activating receptors and the capacity to produce IFN-γ and to degranulate. These data suggest that therapy with HDC plus IL-2 supports the reconstitution of a deficient NK cell fraction through the specific amplification of CD56bright NK cells giving rise to a functional NK cell compartment with high potential to combat leukemic disease.

Drug-induced inhibition of phosphorylation of STAT5 overrides drug resistance in neoplastic mast cells

Systemic mastocytosis (SM) is a mast cell (MC) neoplasm with complex pathology and a variable clinical course. In aggressive SM (ASM) and MC leukemia (MCL), responses to conventional drugs are poor and the prognosis is dismal. R763 is a multi-kinase inhibitor that blocks the activity of Aurora-kinase-A/B, ABL1, AKT and FLT3. We examined the effects of R763 on proliferation and survival of neoplastic MC. R763 produced dose-dependent inhibition of proliferation in the human MC lines HMC-1.1 (IC50 5–50 nM), HMC-1.2 (IC50 1–10 nM), ROSAKIT WT (IC50 1–10 nM), ROSAKIT D816V (IC50 50–500 nM) and MCPV-1.1 (IC50 100–1000 nM). Moreover, R763 induced growth inhibition in primary neoplastic MC in patients with ASM and MCL. Growth-inhibitory effects of R763 were accompanied by signs of apoptosis and a G2/M cell cycle arrest. R763 also inhibited phosphorylation of KIT, BTK, AKT and STAT5 in neoplastic MC. The most sensitive target appeared to be STAT5. In fact, tyrosine phosphorylation of STAT5 was inhibited by R763 at 10 nM. At this low concentration, R763 produced synergistic growth-inhibitory effects on neoplastic MC when combined with midostaurin or dasatinib. Together, R763 is a novel promising multi-kinase inhibitor that blocks STAT5 activation and thereby overrides drug-resistance in neoplastic MC.

The pan-BCL-2-blocker obatoclax (GX15-070) and the PI3-kinase/mTOR-inhibitor BEZ235 produce cooperative growth-inhibitory effects in ALL cells

Acute lymphoblastic leukemia (ALL) is characterized by leukemic expansion of lymphoid blasts in hematopoietic tissues. Despite improved therapy only a subset of patients can be cured. Therefore, current research is focusing on new drug-targets. Members of the BCL-2 family and components of the PI3-kinase/mTOR pathway are critically involved in the regulation of growth and survival of ALL cells. We examined the effects of the pan-BCL-2 blocker obatoclax and the PI3-kinase/mTOR-inhibitor BEZ235 on growth and survival of ALL cells. In 3H-thymidine uptake experiments, both drugs suppressed the in vitro proliferation of leukemic cells in all patients with Philadelphia chromosome-positive (Ph+) ALL and Ph- ALL (obatoclax IC50: 0.01-5 μM; BEZ235, IC50: 0.01-1 μM). Both drugs were also found to produce growth-inhibitory effects in all Ph+ and all Ph- cell lines tested. Moreover, obatoclax and BEZ235 induced apoptosis in ALL cells. In drug-combination experiments, obatoclax and BEZ235 exerted synergistic growth-inhibitory effects on ALL cells. Finally, we confirmed that ALL cells, including CD34+/CD38- stem cells and all cell lines express transcripts for PI3-kinase, mTOR, BCL-2, MCL-1, and BCL-xL. Taken together, this data shows that combined targeting of the PI3-kinase/mTOR-pathway and BCL-2 family-members is a potent approach to counteract growth and survival of ALL cells.Acute lymphoblastic leukemia (ALL) is characterized by leukemic expansion of lymphoid blasts in hematopoietic tissues. Despite improved therapy only a subset of patients can be cured. Therefore, current research is focusing on new drug-targets. Members of the BCL-2 family and components of the PI3-kinase/mTOR pathway are critically involved in the regulation of growth and survival of ALL cells. We examined the effects of the pan-BCL-2 blocker obatoclax and the PI3-kinase/mTOR-inhibitor BEZ235 on growth and survival of ALL cells. In 3H-thymidine uptake experiments, both drugs suppressed the in vitro proliferation of leukemic cells in all patients with Philadelphia chromosome-positive (Ph+) ALL and Ph− ALL (obatoclax IC50: 0.01-5 μM; BEZ235, IC50: 0.01-1 μM). Both drugs were also found to produce growth-inhibitory effects in all Ph+ and all Ph− cell lines tested. Moreover, obatoclax and BEZ235 induced apoptosis in ALL cells. In drug-combination experiments, obatoclax and BEZ235 exerted synergistic growth-inhibitory effects on ALL cells. Finally, we confirmed that ALL cells, including CD34+/CD38− stem cells and all cell lines express transcripts for PI3-kinase, mTOR, BCL-2, MCL-1, and BCL-xL. Taken together, this data shows that combined targeting of the PI3-kinase/mTOR-pathway and BCL-2 family-members is a potent approach to counteract growth and survival of ALL cells.