Phenotyping and Target Expression Profiling of CD34+/CD38- and CD34+/CD38+ Stem- and Progenitor cells in Acute Lymphoblastic Leukemia.

Leukemic stem cells (LSCs) are an emerging target of curative anti-leukemia therapy. In acute lymphoblastic leukemia (ALL), LSCs frequently express CD34 and often lack CD38. However, little is known about markers and targets expressed in ALL LSCs. We have examined marker- and target expression profiles in CD34+/CD38- LSCs in patients with Ph+ ALL (n = 22) and Ph- ALL (n = 27) by multi-color flow cytometry and qPCR. ALL LSCs expressed CD19 (B4), CD44 (Pgp-1), CD123 (IL-3RA), and CD184 (CXCR4) in all patients tested. Moreover, in various subgroups of patients, LSCs also displayed CD20 (MS4A1) (10/41 = 24%), CD22 (12/20 = 60%), CD33 (Siglec-3) (20/48 = 42%), CD52 (CAMPATH-1) (17/40 = 43%), IL-1RAP (13/29 = 45%), and/or CD135 (FLT3) (4/20 = 20%). CD25 (IL-2RA) and CD26 (DPPIV) were expressed on LSCs in Ph+ ALL exhibiting BCR/ABL1p210, whereas in Ph+ ALL with BCR/ABL1p190, LSCs variably expressed CD25 but did not express CD26. In Ph- ALL, CD34+/CD38- LSCs expressed IL-1RAP in 6/18 patients (33%), but did not express CD25 or CD26. Normal stem cells stained negative for CD25, CD26 and IL-1RAP, and expressed only low amounts of CD52. In xenotransplantation experiments, CD34+/CD38- and CD34+/CD38+ cells engrafted NSG mice after 12-20 weeks, and targeting with antibodies against CD33 and CD52 resulted in reduced engraftment. Together, LSCs in Ph+ and Ph- ALL display unique marker- and target expression profiles. In Ph+ ALL with BCR/ABL1p210, the LSC-phenotype closely resembles the marker-profile of CD34+/CD38- LSCs in chronic myeloid leukemia, confirming the close biologic relationship of these neoplasms. Targeting of LSCs with specific antibodies or related immunotherapies may facilitate LSC eradication in ALL.

The JAK2/STAT5 signaling pathway as a potential therapeutic target in canine mastocytoma.

BACKGROUND: Mastocytoma are frequently diagnosed cutaneous neoplasms in dogs. In non-resectable mastocytoma patients, novel targeted drugs are often applied. The transcription factor STAT5 has been implicated in the survival of human neoplastic mast cells (MC). Our study evaluated the JAK2/STAT5 pathway as a novel target in canine mastocytoma. MATERIALS AND METHODS: We employed inhibitors of JAK2 (R763, TG101348, AZD1480, ruxolitinib) and STAT5 (pimozide, piceatannol) and evaluated their effects on 2 mastocytoma cell lines, C2 and NI-1. RESULTS: Activated JAK2 and STAT5 were detected in both cell lines. The drugs applied were found to inhibit proliferation and survival in these cells with the following rank-order of potency: R763 > TG101348 > AZD1480 > pimozide > ruxolitinib > piceatannol. Moreover, synergistic anti-neoplastic effects were obtained by combining pimozide with KIT-targeting drugs (toceranib, masitinib, nilotinib, midostaurin) in NI-1 cells. CONCLUSION: The JAK2/STAT5 pathway is a novel potential target of therapy in canine mastocytoma.

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.

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells.

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.

Evaluation of efficacy of alemtuzumab in 5 patients with aplastic anemia and/or myelodysplastic neoplasm.

Patients with aplastic anemia or hypoplastic myelodysplastic syndrome (MDS) may respond to immunosuppressive therapy, including the anti-CD52 antibody alemtuzumab. We analyzed treatment responses to alemtuzumab in 5 patients with MDS or aplastic anemia (AA) evolving to MDS. Two patients with hypoplastic MDS (hMDS) showed a partial response (PR) to alemtuzumab. In both responding patients, a concomitant paroxysmal nocturnal hemoglobinuria (PNH) clone was detected before therapy. One responder relapsed after 15 months and underwent successful allogeneic stem cell transplantation. Both patients are still alive and in remission after 40 and 20 months, respectively. The other patients showed no response to alemtuzumab. One patient died from pneumonia 4 months after treatment. In summary, our data confirm that alemtuzumab is an effective treatment option for a subset of patients with MDS, even in the presence of a PNH clone.

Maintenance therapy with histamine plus IL-2 induces a striking expansion of two CD56bright 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.

Normal ABL1 is a tumor suppressor and therapeutic target in human and mouse leukemias expressing oncogenic ABL1 kinases.

Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.

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 cellsin vitroand their engraftmentin vivoin 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.

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.

Transcriptional plasticity promotes primary and acquired resistance to BET inhibition.

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

Cancer stem cells in basic science and in translational oncology: can we translate into clinical application?

Since their description and identification in leukemias and solid tumors, cancer stem cells (CSC) have been the subject of intensive research in translational oncology. Indeed, recent advances have led to the identification of CSC markers, CSC targets, and the preclinical and clinical evaluation of the CSC-eradicating (curative) potential of various drugs. However, although diverse CSC markers and targets have been identified, several questions remain, such as the origin and evolution of CSC, mechanisms underlying resistance of CSC against various targeted drugs, and the biochemical basis and function of stroma cell-CSC interactions in the so-called 'stem cell niche.' Additional aspects that have to be taken into account when considering CSC elimination as primary treatment-goal are the genomic plasticity and extensive subclone formation of CSC. Notably, various cell fractions with different combinations of molecular aberrations and varying proliferative potential may display CSC function in a given neoplasm, and the related molecular complexity of the genome in CSC subsets is considered to contribute essentially to disease evolution and acquired drug resistance. In the current article, we discuss new developments in the field of CSC research and whether these new concepts can be exploited in clinical practice in the future.

Long-term treatment with imatinib results in profound mast cell deficiency in Ph+ chronic myeloid leukemia.

Although mast cells (MC) play an important role in allergic reactions, their physiologic role remains unknown. In mice, several models of MC-deficiency have been developed. However, no comparable human model is available. We examined the in vitro- and in vivo effects of the KIT-targeting drug imatinib on growth and development of human MC. Imatinib was found to inhibit stem cell factor (SCF)-induced differentiation of MC in long-term suspension cultures (IC50: 0.01 µM). Correspondingly, long-term treatment of chronic myeloid leukemia (CML) patients with imatinib (400 mg/day) resulted in a marked decrease in MC. In patients with continuous complete molecular response during therapy, bone marrow MC decreased to less than 5% of pre-treatment values, and also serum tryptase concentrations decreased significantly (pre-treatment: 32.0 ± 11.1 ng/ml; post-therapy: 3.4 ± 1.8, p<0.01). Other myeloid lineages, known to develop independently of KIT, were not affected by imatinib-therapy. Imatinib also produced a substantial decrease in MC-development in mice. However, no clinical syndrome attributable to drug-induced MC-deficiency was recorded in our CML patients. Together, imatinib suppresses MC production in vitro and in vivo. However, drug-induced MC depletion is not accompanied by adverse clinical events, suggesting that MC are less relevant to homeostasis in healthy tissues than we assumed so far.

Chronic mast cell leukemia (MCL) with KIT S476I: a rare entity defined by leukemic expansion of mature mast cells and absence of organ damage.

Mast cell leukemia (MCL) is a rare, life-threatening malignancy defined by a substantial increase in neoplastic mast cells (MCs) in bone marrow (BM) smears, drug-resistance, and a poor prognosis. In most patients, the survival time is less than 1 year. However, exceptional cases may present with a less malignant course. We report on a 49-year-old female patient with MCL diagnosed in 2013. In February 2013, first symptoms, including flushing, headache, and diarrhea, were recorded. In addition, mild anemia was detected. The disease was characterized by a massive increase in well-granulated, mature, and often spindle-shaped MCs (80 %) in BM smears. The serum tryptase level amounted to 332 ng/mL. Like in most other MCL patients, no skin lesions were detected. However, unlike in other patients, tryptase levels remained stable, and no other signs or symptoms of MCL-induced organ damage were found. Sequencing studies revealed an isolated S476I point mutation in KIT but no mutation in codon 816. The patient received histamine receptor blockers but refused cytoreductive therapy. After 9 months, still no progression or organ damage was detected. However, progression with transformation to acute MCL occurred after 12 months. We propose that the chronic type of MCL with stable conditions, absence of organ damage, and a mature MC morphology is recognized as a distinct entity that should be distinguished from the acute variant of MCL.

DPPIV (CD26) as a novel stem cell marker in Ph+ chronic myeloid leukaemia.

The concept of leukaemic stem cells (LSCs) has been developed to explain the complex cellular hierarchy and biology of leukaemias and to screen for pivotal targets that can be employed to improve drug therapies through LSC eradication in these patients. Some of the newly discovered LSC markers seem to be expressed in a disease-specific manner and may thus serve as major research tools and diagnostic parameters. A useful LSC marker in chronic myeloid leukaemia (CML) appears to be CD26, also known as dipeptidylpeptidase IV. Expression of CD26 is largely restricted to CD34(+) /CD38(-) LSCs in BCR/ABL1(+) CML, but is not found on LSCs in other myeloid or lymphoid neoplasms, with the exception of lymphoid blast crisis of CML, BCR/ABL1p210 + acute lymphoblastic leukaemia, and a very few cases of acute myeloid leukaemia. Moreover, CD26 usually is not expressed on normal bone marrow (BM) stem cells. Functionally, CD26 is a cytokine-targeting surface enzyme that may facilitate the mobilization of LSCs from the BM niche. In this article, we review our current knowledge about the biology and function of CD26 on CML LSCs and discuss the diagnostic potential of this new LSC marker in clinical haematology.

Identification of campath-1 (CD52) as novel drug target in neoplastic stem cells in 5q-patients with MDS and AML.

PURPOSE: The CD52-targeted antibody alemtuzumab induces major clinical responses in a group of patients with myelodysplastic syndromes (MDS). The mechanism underlying this drug effect remains unknown. EXPERIMENTAL DESIGN: We asked whether neoplastic stem cells (NSC) in patients with MDS (n = 29) or acute myelogenous leukemia (AML; n = 62) express CD52. RESULTS: As assessed by flow cytometry, CD52 was found to be expressed on NSC-enriched CD34(+)/CD38(-) cells in 8/11 patients with MDS and isolated del(5q). In most other patients with MDS, CD52 was weakly expressed or not detectable on NSC. In AML, CD34(+)/CD38(-) cells displayed CD52 in 23/62 patients, including four with complex karyotype and del(5q) and one with del(5q) and t(1;17;X). In quantitative PCR (qPCR) analyses, purified NSC obtained from del(5q) patients expressed CD52 mRNA. We were also able to show that CD52 mRNA levels correlate with EVI1 expression and that NRAS induces the expression of CD52 in AML cells. The CD52-targeting drug alemtuzumab, was found to induce complement-dependent lysis of CD34(+)/CD38(-)/CD52(+) NSC, but did not induce lysis in CD52(-) NSC. Alemtuzumab also suppressed engraftment of CD52(+) NSC in NSG mice. Finally, CD52 expression on NSC was found to correlate with a poor survival in patients with MDS and AML. CONCLUSIONS: The cell surface target Campath-1 (CD52) is expressed on NSC in a group of patients with MDS and AML. CD52 is a novel prognostic NSC marker and a potential NSC target in a subset of patients with MDS and AML, which may have clinical implications and may explain clinical effects produced by alemtuzumab in these patients.

CD52 is a molecular target in advanced systemic mastocytosis.

Advanced systemic mastocytosis (SM) is an aggressive hematopoietic neoplasm with poor prognosis and short survival times. So far, no curative therapy is available for affected patients. We have identified the cell surface antigen CD52 (CAMPATH-1) as a molecular target expressed abundantly on the surface of primary neoplastic mast cells (MCs) in patients with advanced SM. In contrast, neoplastic MCs of patients with indolent SM and normal MCs expressed only low levels or did not express CD52. To study the mechanisms of CD52 expression and the value of this antigen as a potential therapeutic target, we generated a human MC cell line, designated MCPV-1, by lentiviral immortalization of cord blood-derived MC progenitor cells. Functional studies revealed that activated RAS profoundly promotes surface expression of CD52. The CD52-targeting antibody alemtuzumab induced cell death in CD52(+) primary neoplastic MCs obtained from patients with SM as well as in MCPV-1 cells. NSG mice xenotransplanted with MCPV-1 cells survived significantly longer after treatment with alemtuzumab (median survival: 31 d untreated vs. 46 d treated; P=0.0012). We conclude that CD52 is a novel marker and potential therapeutic target in neoplastic MCs in patients with advanced SM.

Identification of heat shock protein 32 (Hsp32) as a novel target in acute lymphoblastic leukemia.

Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in oncology. We have shown that Hsp32, also known as heme oxygenase-1 (HO-1), serves as survival factor and potential target in Ph+ chronic myeloid leukemia. We here report that primary cells and cell lines derived from patients with acute lymphoblastic leukemia (ALL) express Hsp32 mRNA and the Hsp32 protein in a constitutive manner. Highly enriched CD34+/CD38- ALL stem cells also expressed Hsp32. Two Hsp32-targeting drugs, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), induced apoptosis and growth arrest in the BCR/ABL1+ cell lines, in Ph- lymphoblastic cell lines and in primary Ph+ and Ph- ALL cells. The effects of PEG-ZnPP and SMA-ZnPP on growth of leukemic cells were dose-dependent. In Ph+ ALL, major growth-inhibitory effects of the Hsp32-targeting drugs were observed in imatinib-sensitive and imatinib-resistant cells. Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells. A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells. In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

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.

FLAG-induced remission in a patient with acute mast cell leukemia (MCL) exhibiting t(7;10)(q22;q26) and KIT D816H.

Mast cell leukemia (MCL) is a life-threatening disease associated with high mortality and drug-resistance. Only few patients survive more than 12 months. We report on a 55-year-old female patient with acute MCL diagnosed in May 2012. The disease was characterized by a rapid increase in white blood cells and mast cells (MC) in the peripheral blood, and a rapid increase of serum tryptase levels. The KIT D816H mutation was detected in the blood and bone marrow (BM). Induction chemotherapy with high-dose ARA-C and fludarabine (FLAG) was administered. Unexpectedly, the patient entered a hematologic remission with almost complete disappearance of neoplastic MC and a decrease of serum tryptase levels to normal range after 2 cycles of FLAG. Consecutively, the patient was prepared for allogeneic stem cell transplantation. However, shortly after the third cycle of FLAG, tryptase levels increased again, immature MC appeared in the blood, and the patient died from cerebral bleeding. Together, this case shows that intensive chemotherapy regimens, like FLAG, may induce remission in acute MCL. However, treatment responses are short-lived and the overall outcome remains dismal in these patients. We propose to separate this acute type of MCL from more subacute or chronic variants of MCL.

Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy.

Chronic myeloid leukemia and systemic mastocytosis are myeloid neoplasms sharing a number of pathogenetic and clinical features. In both conditions, an aberrantly activated oncoprotein with tyrosine kinase activity, namely BCR-ABL1 in chronic myeloid leukemia, and mutant KIT, mostly KIT D816V, in systemic mastocytosis, is key to disease evolution. The appreciation of the role of such tyrosine kinases in these diseases has led to the development of improved therapies with tyrosine kinase-targeted inhibitors. However, most drugs, including new KIT D816V-blocking agents, have failed to achieve long-lasting remissions in advanced systemic mastocytosis, and there is a similar problem in chronic myeloid leukemia, where imatinib-resistant patients sometimes fail to achieve remission, even with second- or third-line BCR-ABL1 specific tyrosine kinase inhibitors. During disease progression, additional signaling pathways become activated in neoplastic cells, but most converge into major downstream networks. Among these, the AKT and STAT5 pathways appear most critical and may result in drug-resistant chronic myeloid leukemia and systemic mastocytosis. Inhibition of phosphorylation of these targets has proven their crucial role in disease-evolution in both malignancies. Together, these observations suggest that STAT5 and AKT are key drivers of oncogenesis in drug-resistant forms of the diseases, and that targeting STAT5 and AKT might be an interesting approach in these malignancies. The present article provides an overview of our current knowledge about the critical role of AKT and STAT5 in the pathophysiology of chronic myeloid leukemia and systemic mastocytosis and on their potential value as therapeutic targets in these neoplasms.