STAT5 transcription factors gain increasing attention as essential drivers in the development of myeloproliferative and lymphoid diseases. STAT5 is often hyper-activated due to deregulated upstream ...kinase signaling. Additionally, recurrent gain-of-function mutations in the SH2 domain of STAT5 were described, implying its importance in oncogenic transformation independent of driver kinase mutations. To date, various tyrosine kinase inhibitors (TKI) are in the clinics or in clinical trials. However, TKI treatment is often accompanied by resistance development, cytotoxicity as a result of poor kinase selectivity, as well as cardiovascular toxicity. Hence, considering the role of STAT5 in hematopoietic cancers, it is reasonable to directly target STAT5 as a transcriptional regulator.
To inhibit STAT5, we used a small inhibitory molecule (compound X) binding to the SH2 domain of the protein, subsequently resulting in the disruption of STAT5-phosphopeptide interactions. We tested the efficacy of X in STAT5 driven AML/CML cell lines and patient samples. The inhibitor efficiently suppressed transcriptional activity of STAT5, kinase-mediated phosphorylation, dimerization, nuclear translocation, DNA binding and STAT5-mediated gene expression. Importantly, we detected down regulation of c-MYC, Cyclin D1, Cyclin D2, and MCL-oncoproteins, which are important inhibitors of apoptosis and regulators of the cell cycle. Furthermore, X induced extensive apoptosis in MPN cell lines as well as robust apoptotic cell death in patient-derived cells.
Our findings indicate that X is a potent and selective inhibitor of STAT5 and provides a lead structure for further chemical modifications for clinical development to improve existing therapies in MPNs.
Leukemic stem cells (LSCs) have recently been identified as an important target of therapy in various human leukemias and related blood cell disorders. Systemic mastocytosis (SM) is a rare ...hematologic neoplasm characterized by abnormal growth and accumulation of mast cells (MCs) in various organ systems, including the bone marrow (BM). Whereas patients with indolent SM (ISM) have a normal life-expectancy, patients with more advanced forms of SM have a poor prognosis. In these patients, neoplastic MCs are usually resistant against conventional drugs and various targeted drugs. MC leukemia (MCL) is the rare leukemic variant of advanced SM, defined by a rapidly devastating expansion of immature MCs in various hematopoietic organs and a poor prognosis with short survival times. Although MCL is considered a stem cell disease, little is known about the origin and phenotype of MCL-initiating LSCs. We examined the phenotypic and functional characteristics of putative LSCs in patients with aggressive SM (ASM, n=12) and MCL (n=6). Putative LSCs were identified and characterized phenotypically by flow cytometry. Highly enriched, sorted LSCs were injected into NOD-SCID-IL-2Rγ-/- mice exhibiting a 220 amino acid isoform of human membrane-bound hSCF (NSGSCF). We found that disease-initiating and propagating LSCs reside within a CD34+ fraction of the MCL clone. Whereas cell fractions containing CD34+ cells as well as highly enriched CD34+ cells produced engraftment in NSGSCF mice with a MCL-like disease (43-77% human MCL cells in mouse BM after 10-22 weeks), no substantial engraftment was produced by MC-rich but stem cell-depleted, KIT+/CD34─ cell fractions obtained from the same patients (<1% engraftment in mouse BM). In dilution experiments, engraftment of CD34+ cells was documented down to a minimum of 50 cells per mouse. The identity of engrafting MCL cells was confirmed by morphology, phenotyping and molecular studies demonstrating the presence of KIT mutations that were initially detected in the primary MCL samples used. Moreover, we were able to confirm long-term engraftment by successful serial transplantations into secondary recipient mice. In consecutive experiments, we were able to show that CD45+/CD34+/CD38─ cells also produce leukemic engraftment in NSGSCF mice. As assessed by flow cytometry, these CD34+/CD38─ MCL LSCs were found to express several stem cells markers, including aminopeptidase-N (CD13), leukosialin (CD43), Pgp-1 (CD44), the IL-3R alpha-chain (CD123), AC133 (CD133) and CXCR4 (CD184). In addition, in most patients examined, MCL LSCs were found to display IL-1RAP, a surface antigen that is otherwise expressed in CML LSCs but is not expressed in normal stem cells. In addition, MCL LSCs were found to express various cell surface targets, including CD33 and CD52. By contrast, MCL LSCs did not express CD2, CD25, CD26 and CLL-1. The more mature progenitor cell fractions (CD34+/CD38+) were found to stain positive for CD13, CD33, CD43, CD44, CD90, CD117, CD123, CD133 and CD184. Mature clonal MCs expressed a similar phenotype, including molecular markers and targets, such as CD13, CD30 CD33, CD52 and CD184. In patients with ISM and aggressive SM (ASM), the CD34+/CD38─ stem cells exhibited a similar surface marker profile compared to MCL, but expressed lower levels of CD133 and did not express IL-1RAP. In the validation phase of our study, we examined the effects of target-specific antibodies. As assessed by flow cytometry, the CD52-targeting antibody alemtuzumab was found to induce complement-dependent lysis of CD34+ and CD34+/CD38─ cells in all MCL samples analysed. Furthermore, pre-incubation of MCL cells with alemtuzumab prior to injection into NSGSCF mice resulted in a significantly reduced engraftment (2.7±4.1%) after 22 weeks. In conclusion, our data show that the MCL clone originates from a primitive hematopoietic stem cell that co-expresses CD34, CD123, CD133 and IL-1RAP but lacks CD25 and CD26. In addition, our data show that MCL LSC express a number of clinically relevant surface targets, including CD33, CD52 and CD117 (KIT). These observations may facilitate LSC detection and isolation in MCL and may lead to the development of novel LSC-eradicating treatment concepts in this highly aggressive and drug-resistant form of leukemia.
Valent:Novartis: Consultancy, Honoraria, Research Funding.
In the past decade, the treatment of chronic myeloid leukemia (CML) has undergone a drastic evolution. The discovery and success of imatinib and second-generation tyrosine kinase inhibitors have ...substantially increased the outcome for CML patients. The next step in medical and scientific research is to better understand the malignancy so as to eventually find a cure to eliminate all leukemic cells from patients. One of the key issues is about the resistance of the leukemic stem cells to tyrosine kinase inhibitors. Here, we briefly describe our current studies on CML resistance, and leukemic stem cell modeling and characterization.
In chronic myeloid leukemia (CML), resistance against second-generation tyrosine kinase inhibitors (TKI) remains a serious clinical challenge, especially in the context of multi-resistant BCR-ABL1 ...mutants, such as T315I. Treatment with ponatinib may suppress most of these mutants, including T315I, but is also associated with a high risk of clinically relevant side effects. We screened for alternative treatment options employing available tyrosine kinase inhibitors (TKI) in combination. Dasatinib and bosutinib are two second-generation TKI that bind to different, albeit partially overlapping, spectra of kinase targets in CML cells. This observation prompted us to explore anti-leukemic effects of the combination dasatinib + bosutinib in highly resistant primary CML cells, various CML cell lines (K562, K562R, KU812, KCL22) and Ba/F3 cells harboring various BCR-ABL1 mutant-forms. We found that bosutinib synergizes with dasatinib in inducing growth inhibition and apoptosis in all CML cell lines and in Ba/F3 cells exhibiting BCR-ABL1
. Clear synergistic effects were also observed in primary CML cells in all patients tested (n = 20), including drug-resistant cells carrying BCR-ABL1
. Moreover, the drug combination produced cooperative or even synergistic apoptosis-inducing effects on CD34
/CD38
CML stem cells. Finally, we found that the drug combination is a potent approach to block the activity of major additional CML targets, including LYN, KIT and PDGFRα. Together, bosutinib and dasatinib synergize in producing anti-leukemic effects in drug-resistant CML cells. Whether such cooperative TKI effects also occur in vivo in patients with drug-resistant CML, remains to be determined in forthcoming studies.
Abstract 1760
In chronic eosinophilic leukemia (CEL), the transforming oncoprotein FIP1L1-PDGFRA (F/P) is a major target of therapy. In most patients, the PDGFRA-targeting tyrosine kinase inhibitor ...(TKI) imatinib induces complete and durable molecular remissions. For patients who are intolerant or resistant against imatinib, novel TKI may serve as potential alternative therapy. Indeed, several different TKI have been described to act on Ba/F3 cells transfected with F/P, and some even block the activity of imatinib-resistant F/P mutants. However, little is known about the effects of novel TKI on growth and survival of primary neoplastic eosinophils. In the current study, we examined the in vitro effects of 12 kinase blockers on growth and viability as well as cytokine-induced migration of EOL-1 cells, a human F/P+ eosinophil leukemia cell line. In addition, we examined TKI effects on primary human neoplastic eosinophils obtained from a patient with F/P+ CEL, one with aggressive systemic mastocytosis and massive eosinophilia (ASM-eo) and one with reactive hypereosinophilia (HE). In EOL-1 cells, major growth-inhibitory effects were seen with all PDGFRA-blocking agents, with IC50 values in the low nM-range: ponatinib: 0.1–0.2 nM, sorafenib: 0.1–0.2 nM, masitinib: 0.2–0.5 nM, nilotinib: 0.2–2 nM, dasatinib: 0.5–2 nM, sunitinib: 1–2 nM, and midostaurin: 5–10 nM. These drugs were also found to block the activity of PDGFR-downstream signaling molecules, including Akt, S6, and STAT5 in EOL-1 cells. Targeting of individual downstream molecules with specific inhibitors (PI3-kinase: NVP-BEZ235; mTOR: everolimus; STAT5: pimozide and piceatannol) also induced growth-inhibition in EOL-1 cells, although IC50 values were higher compared to that obtained with PDGFR-blocking TKI. All effective TKI produced dose-dependent apoptosis in EOL-1 cells as determined by microscopy, Annexin-V/PI staining, and staining for active caspase-3. In a next step, we applied the most effective TKI on primary neoplastic eosinophils. In these experiments, ponatinib, dasatinib, and nilotinib were found to suppress the growth of primary neoplastic eosinophils obtained from a patient with F/P+ CEL and one with ASM-eo, in a dose-dependent manner (IC50 <0.5 μM). In the patient with reactive HE, the TKI also produced growth inhibition, but IC50 values were higher compared to neoplastic eosinophils. We also examined drug effects on growth of Ba/F3 cells expressing the imatinib-resistant F/P mutants T674I and D842V. In these experiments, sunitinib was found to inhibit the growth of Ba/F3 cells expressing the T674I mutant of F/P. By contrast, no substantial effects of masitinib or nilotinib on Ba/F3 cells expressing this mutant were found, and Ba/F3 cells expressing F/P D842V were found to be resistant against sunitinib and masitinib. Strong inhibitory effects on both mutants were only seen with ponatinib. We next examined the effects of various TKI on cytokine-induced migration of neoplastic eosinophils. Unexpectedly, of all cytokines tested including IL-5 and eotaxin, only SDF-1A was found to induce in vitro migration of EOL-1 cells. We found that imatinib, nilotinib, dasatinib, ponatinib, sorafenib, and masitinib inhibit SDF-1A-induced migration of EOL-1 cells in a dose-dependent manner (effective range: 10–100 nM). Finally, we analyzed TKI effects on expression of activation-linked cell surface antigens on EOL-1 cells. In these experiments, we found that ponatinib and sorafenib downregulate expression of CD25 and CD63 in EOL-1 cells, whereas the other TKI tested showed no effects. By contrast, no effects of ponatinib or sorafenib on expression of HLA-DR, CXCR4 and CD95 on EOL-1 cells were seen. We were also unable to detect any significant effects of the other TKI on expression of activation-linked cell surface antigens in EOL-1 cells. In summary, our data show that various novel TKI counteract growth, survival, activation, and migration of neoplastic human eosinophils. The most potent agent that also blocks all known mutant-forms of F/P appears to be ponatinib. Novel PDGFR-targeting TKI, such as ponatinib, may be attractive alternative drugs for the treatment of imatinib-resistant or intolerant CEL.
Valent:Phadia: Research Funding.
Chronic myeloid leukemia (CML) is a stem cell neoplasm characterized by the Philadelphia (Ph) chromosome and the related oncoprotein, BCR/ABL. Despite the availability of novel BCR/ABL-targeting ...tyrosine kinase inhibitors (TKI), many patients relapse which may be due to resistance of neoplastic stem cells (NSC) against TKI. So far, little is known about specific markers and targets expressed on CML NSC. We here report that CML NSC express the interleukin-2R alpha-chain CD25 in a specific (aberrant) manner. Whereas normal bone marrow (BM) stem cells were found to express only low amounts or did not express CD25, CD34+/CD38− NSC in CML were found to express CD25 in almost all patients examined (31/33=95%), independent of the phase of disease. CML NSC were also found to express IL-1RAP, DPPIV (CD26), Siglec-3 (CD33), Campath-1 (CD52), KIT (CD117) and IL-3RA (CD123). We were also able to show that highly purified CD25+ NSC express BCR/ABL and engraft irradiated NOD-SCID-IL-2Rg-/- (NSG) mice with BCR/ABL+ cells, whereas CD34+/CD38-/CD25-/CD26- cells from the same patients failed to express BCR/ABL, and engrafted NSG mice with BCR/ABL-negative cells. To define signalling-molecules contributing to the expression of CD25 in CML NSC, we employed primary murine hematopoietic cells infected with bcr/abl-p210 in combination with a retrovirus encoding for STAT5A or STAT5B. Enforced expression of either STAT5A or STAT5B resulted in enhanced expression of CD25 in Lin-/Sca-1+/Kit+ mouse BM stem cells (LSK cells), regardless of the presence or absence of bcr/abl. In fact, the CD25-promoting effect of STAT5 was seen in normal stem cells as well as in bcr/abl-transformed LSK cells. Correspondingly, shRNA against STAT5 was found to downregulate expression of CD25 in the human CML cell line KU812. The BCR/ABL-targeting TKI imatinib, nilotinib and ponatinib were also found to inhibit expression of CD25 on KU812 cells. In primary CML NSC, ponatinib was found to induce marked suppression of CD25, whereas imatinib and nilotinib showed only weak effects. In consecutive experiments, we found that the MEK inhibitors RDEA119 and PD032509, the STAT5-targeting drug pimozide and the dual PI3 kinase/mTOR blocker BEZ235 all inhibit 3H-thymidine uptake and thus proliferation in KU812 cells. However, whereas RDEA119 and PD032509 as well as pimozide were found to downregulate expression of CD25 in KU812 cells, BEZ235 was found to even upregulate expression of CD25 on KU812 cells. We next applied an shRNA against CD25. The shRNA-induced knock-down of CD25 in KU812 resulted in an increased proliferative capacity, suggesting that CD25 may act as a potential tumor suppressor in CML stem- and progenitor cells. Together, our data show that CD25 is a novel functionally relevant marker-antigen and potential tumor suppressor of CML NSC. Expression of CD25 in CML cells appears to be triggered by MEK and STAT5 activity, and may be negatively regulated by the PI3 kinase/mTOR pathway. Whether modulation of CD25 can be exploited clinically and used as basis for the development of novel NSC-targeting treatment concepts in CML remains to be determined in future studies.
Valent:Novartis: Consultancy, Honoraria, Research Funding.
Abstract 2280
In most patients with chronic myeloid leukemia (CML), complete cytogenetic remission can be achieved with the BCR/ABL tyrosine kinase inhibitor (TKI) imatinib. However, not all patients ...are long-term responders. A major cause of acquired resistance against imatinib is the development of BCR/ABL mutations in subclones. In most of these patients, a second generation TKI is prescribed. However, the T315I mutant of BCR/ABL introduces resistance against most TKI, including nilotinib and dasatinib. One approach to overcome drug resistance in BCR/ABL T315I+ CML cells may be to apply drug combinations. Recent data suggest that the mechanisms through which dasatinib and nilotinib act on BCR/ABL differ from each other and that both drugs act on multiple additional targets in CML cells. Here, we show that dasatinib and nilotinib cooperate with each other in producing growth inhibition in imatinib-sensitive and imatinib-resistant CML cells, including subclones bearing BCR/ABL T315I. The drug combination was tested on leukemic cells obtained from 9 patients with chronic phase (CP) CML and 3 with blast phase (PB) of CML. Samples were assessed from 4 patients at the time of diagnosis, and against cells from 8 patients (CP, n=5; BP, n=3) who had developed resistance against one or more BCR/ABL TKI. In all 3 patients in PB, the T315I mutant was detectable. As expected, nilotinib and dasatinib failed to inhibit proliferation of cells harbouring BCR/ABL T315I when applied as single agents. However, the combination xnilotinib+dasatinibx produced synergistic effects in most samples, including primary CML cells and Ba/F3 cells harbouring BCR/ABL T315I. Interestingly, in all 3 patients with BP (BCR/ABL T315I+), strong cooperative or even synergistic growth-inhibitory effects were observed in primary CML cells, resulting in substantial anti-leukemic effects seen at reasonable (pharmacologic) drug concentrations (< 1 μ M) (figure). Based on these results, we treated one patient with TKI-resistant CML in hematologic relapse in whom 2 BCR/ABL mutant-bearing subclones, one clinically resistant against nilotinib (F359V) and one clinically resistant against dasatinib (F317L) had been detected, with a combination of nilotinb (800 mg p.o. daily) and dasatinib (50 mg/day p.o., days 1–5 every third week). A transient hematologic response was obtained in this patient, and except for mild bone pain, no side effects were recorded. Moreover, we were able to show that during treatment with xnilotinib+dasatinibx, the number of CD34+/CD38-/CD33+ CML stem cells decreased from clearly measurable levels (0.005%) to nearly undetectable levels (0.0002%). Finally, ex vivo analyses of leukemic blood cells confirmed, that the combination xnilotinib+dasatinibx produced strong cooperative growth-inhibitory effects in both disease-components, i.e. the F359V-bearing subclone and the F317L-bearing subclone. In summary, our data show that the combination of dasatinib and nilotinib can override acquired TKI resistance in CML, and can suppress growth of various imatinib-resistant subclones including cells that bear BCR/ABL T315I or other BCR/ABL mutants. Whether this combination can suppress imatinib-resistant subclones in CML for prolonged time periods or even can eradicate neoplastic stem cells remains in CML patients to be determined.
Synergistic effects of nilotinib and dasatinib on primary leukemic cells obtained from a patient with a BCR/ABL T315I+ blast phase of CML
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Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.
Nilotinib, an inhibitor of BCR/ABL1 is increasingly used to treat patients suffering from chronic myeloid leukemia (CML). However, treatment with nilotinib is associated with the occurrence of ...vascular adverse events, including progressive atherosclerosis resulting in arterial occlusive disease (AOD). We have recently shown that these events are recurrent and severe and accumulate over time in our patients (n=36). The percentage of patients developing i) one or more clinically overt (symptomatic) AOD-related events and ii) one or more clinically severe AOD events requiring surgical intervention and/or prolonged hospitalization increased from 26.5% and 17.6% at a median observation time (MOT) of 24 months to 44.4% and 19.4% after a MOT of 44 months. The frequency of AOD was lower in all control cohorts examined, including risk factor- and age-matched CML patients treated with imatinib, patients suffering from myelodysplastic syndromes, patients with JAK2-mutated myeloproliferative neoplasms and patients with lymphoid neoplasms. In order to explore the potential pro-atherogenic potential of nilotinib, we employed ApoE knock-out mice. In these mice, treatment with nilotinib (75 mg/kg/day p.o. for 8 weeks) was found to promote plaque formation and thus atherosclerosis when compared to control-mice (percent aortic plaque-area: vehicle-control: 16±3.2%; imatinib: 19.4±8.2%; nilotinib: 22.4±2.9%; p<0.005 for nilotinib vs control). To evaluate the effects of nilotinib on vascular repair processes following stenosis, we also employed a mouse model of hindlimb ischemia. Here, nilotinib (75 mg/kg/day p.o. for 28 days) was found to decrease reperfusion after induction of ischemia whereas imatinib (100 mg/kg/day p.o. for 28 days) showed no comparable effect as determined by laser Doppler perfusion imaging. The decreased perfusion seen in the nilotinib-treated mice was accompanied by an increased rate of limb necrosis as well as a decrease in microvessel density when compared to imatinib-treated mice or control-mice (p<0.05). In addition, we found that the pro-atherogenic cytoadhesion molecule VCAM-1 is expressed at higher levels in vascular cells in nilotinib-treated mice compared to imatinib-treated mice or control animals (cells/high power field: control: 13.9±11.9; imatinib: 14.9±11; nilotinib: 22.6±9.3). In a next step, we examined the in vitro effects of nilotinib on cultured human umbilical vein endothelial cells (HUVEC). In these experiments, we found that nilotinib (between 1-10 µM), but not imatinib (1-10 µM) promotes the expression of the cytoadhesion molecules ICAM-1, VCAM-1 and E-Selectin on HUVEC, confirming our data obtained in mice. In addition, we were able to confirm the anti-angiogenic effects of nilotinib seen in mice by our in vitro experiments. In particular, nilotinib was found to inhibit the migration of HUVEC in a wound-scratch assay as well as angiogenesis in a tube-formation assay whereas imatinib showed no comparable effect. Moreover, nilotinib was found to inhibit the proliferation of HUVEC in a dose-dependent manner (IC50: 1.0 µM) whereas imatinib showed no substantial effect up to 5 µM. Finally, we examined bone marrow (BM) microvessel density in 8 CML patients before and during treatment with nilotinib (800 mg/day for at least 1 year). In these experiments, we found that the numbers of CD34+ endothelial cells per high power field decreases substantially during treatment with nilotinib (before nilotinib: 12.3±2 versus post-nilotinib: 5.5±2.6, p<0.05). In summary, nilotinib exerts direct proatherogenic and growth-inhibitory and thus anti-angiogenic effects on vascular endothelial cells. Whether these effects contribute to nilotinib-associated vasculopathy in patients with CML is currently under investigation.
Wolf:BMS: Speakers Bureau; Pfizer: Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Valent:Pfizer: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Consultancy, Honoraria, Research Funding.
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