Alice M.S. Cheung

Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential.

Aldehyde dehydrogenase (ALDH) activity is used to define normal hematopoietic stem cell (HSC), but its link to leukemic stem cells (LSC) in acute myeloid leukemia (AML) is currently unknown. We hypothesize that ALDH activity in AML might be correlated with the presence of LSC. Fifty-eight bone marrow (BM) samples were collected from AML (n=43), acute lymphoblastic leukemia (ALL) (n=8) and normal cases (n=7). In 14 AML cases, a high SSCloALDHbr cell population was identified (ALDH+AML) (median: 14.89%, range: 5.65–48.01%), with the majority of the SSCloALDHbr cells coexpressing CD34+. In another 29 cases, there was undetectable (n=23) or rare (⩽5%) (n=6) SSCloALDHbr population (ALDH−AML). Among other clinicopathologic variables, ALDH+AML was significantly associated with adverse cytogenetic abnormalities. CD34+ BM cells from ALDH+AML engrafted significantly better in NOD/SCID mice (ALDH+AML: injected bone 21.11±9.07%; uninjected bone 1.52±0.75% vs ALDH−AML: injected bone 1.77±1.66% (P=0.05); uninjected bone 0.23±0.23% (P=0.03)) with the engrafting cells showing molecular and cytogenetic aberrations identical to the original clones. Normal BM contained a small SSCloALDHbr population (median: 2.92%, range: 0.92–5.79%), but none of the ALL cases showed this fraction. In conclusion, SSCloALDHbr cells in ALDH+AML might denote primitive LSC and confer an inferior prognosis in patients.

Sorafenib treatment of FLT3-ITD+ acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent nonresponsiveness associated with the emergence of a D835 mutation

Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% of acute myeloid leukemias (AMLs) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD(+) AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21-84) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54-287) days but the FLT3 and downstream effectors remained suppressed. Gene expression profiling showed that leukemia cells that have become sorafenib resistant expressed several genes including ALDH1A1, JAK3, and MMP15, whose functions were unknown in AML. Nonobese diabetic/severe combined immunodeficiency mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain mutations at D835 were identified in leukemia initiating cells (LICs) from samples before sorafenib treatment. LICs bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggest that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations, and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors.

Enhanced normal short-term human myelopoiesis in mice engineered to express human-specific myeloid growth factors

UNLABELLED Better methods to characterize normal human hematopoietic cells with short-term repopulating activity cells (STRCs) are needed to facilitate improving recovery rates in transplanted patients.We now show that 5-fold more human myeloid cells are produced in sublethally irradiated NOD/SCID-IL-2Receptor-γchain-null (NSG) mice engineered to constitutively produce human interleukin-3, granulocyte-macrophage colony-stimulating factor and Steel factor (NSG-3GS mice) than in regular NSG mice 3 weeks after an intravenous injection of CD34 human cord blood cells. Importantly, the NSG-3GS mice also show a concomitant and matched increase in circulating mature human neutrophils. Imaging NSG-3GS recipients of lenti-luciferase-transduced cells showed that human cells being produced 3 weeks posttransplant were heterogeneously distributed, validating the blood as a more representative measure of transplanted STRC activity. Limiting dilution transplants further demonstrated that the early increase in human granulopoiesis in NSG-3GS mice reflects an expanded output of differentiated cells per STRC rather than an increase in STRC detection. KEY POINTS NSG-3GS mice support enhanced clonal outputs from human short-term repopulating cells (STRCs) without affecting their engrafting efficiency. Increased human STRC clone sizes enable their more precise and efficient measurement by peripheral blood monitoring.

Stem cell model of hematopoiesis.

Hematopoietic stem cells (HSC) are characterized by their capacity of self-renewal, multi-lineage differentiation, and the ability to rescue lethally irradiated hosts. Both murine and human studies have attempted to characterize and purify HSC based on surface phenotypes, metabolic markers, in-vitro clonogenic and in-vivo competitive repopulation assays. The cell-fate of HSC is under intrinsic regulation by various transcription factors, including Hox and SCL genes, cyclin-dependent kinase inhibitors and telomerase, and extrinsic regulation by various signaling pathways involved in embryonic development, including the Notch, Wnt and bone morphogenetic proteins (BMP) pathways. Recent advances in genome research and gene profiling technologies have begun to unravel the regulatory mechanism of HSC by novel genes with hitherto unknown functions in hematopoiesis. The stem cell model of hematopoiesis has also shed light on the concepts of leukemic stem cells (LSC), which involves the presence of a rare population of cells that share the essential HSC attributes of self-renewing, replication and differentiation into progenies of leukemic blasts.

Homoharringtonine (omacetaxine mepesuccinate) as an adjunct for FLT3-ITD acute myeloid leukemia.

An in vitro drug screening using primary AML samples identified homoharringtonine (omacetaxine mepesuccinate) as an effective adjunct for treatment of FLT3-ITD AML. More than a FLT-ing success in leukemia Acute myeloid leukemia is a difficult disease to treat under the best of circumstances, and the subtype containing internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) tends to be particularly challenging. Lam et al. performed a high-throughput drug screen and identified homoharringtonine as a candidate treatment for this type of leukemia and then confirmed its effectiveness in cancer cells, in mouse models, and in patients. The treatment showed promising results in a phase 2 clinical trial, which included elderly patients and those who have failed all previous treatments, paving the way for further development of this drug. An in vitro drug-screening platform on patient samples was developed and validated to design personalized treatment for relapsed/refractory acute myeloid leukemia (AML). Unbiased clustering and correlation showed that homoharringtonine (HHT), also known as omacetaxine mepesuccinate, exhibited preferential antileukemia effect against AML carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD). It worked synergistically with FLT3 inhibitors to suppress leukemia growth in vitro and in xenograft mouse models. Mechanistically, the effect was mediated by protein synthesis inhibition and reduction of short-lived proteins, including total and phosphorylated forms of FLT3 and its downstream signaling proteins. A phase 2 clinical trial of sorafenib and HHT combination treatment in FLT3-ITD AML patients resulted in complete remission (true or with insufficient hematological recovery) in 20 of 24 patients (83.3%), reduction of ITD allelic burden, and median leukemia-free and overall survivals of 12 and 33 weeks. The regimen has successfully bridged five patients to allogeneic hematopoietic stem cell transplantation and was well tolerated in patients unfit for conventional chemotherapy, including elderly and heavily pretreated patients. This study validated the principle and clinical relevance of in vitro drug testing and identified an improved treatment for FLT3-ITD AML. The results provided the foundation for phase 2/3 clinical trials to ascertain the clinical efficacy of FLT3 inhibitors and HHT in combination.

FLT3/internal tandem duplication subclones in acute myeloid leukemia differ in their engraftment potential in NOD/SCID mice.

In this study, we tested if FLT3/internal tandem duplication (ITD) in acute myeloid leukemia (AML) might occur at different hierarchical stages during leukemogenesis. In 56 AML cases, 10 showed FLT3/ITD (single ITD=5; multiple ITD=5). Myeloblasts from seven cases (CD34-selected=4; unselected=3) were transplanted into NOD/SCID mice. Five cases engrafted successfully into 14 mice. Two patients carried single FLT3/ITD subclones, which were maintained during primary and secondary transplantations. In three patients with multiple FLT3/ITD subclones, some subclones persisted or expanded while others diminished upon transplantation. Their different engraftment capabilities in NOD/SCID mice supported the proposition that FLT3/ITD might occur at different stages during leukemogenesis.

Successful engraftment by leukemia initiating cells in adult acute lymphoblastic leukemia after direct intrahepatic injection into unconditioned newborn NOD/SCID mice

OBJECTIVE Xenogeneic transplantation has been the gold standard for enumeration of leukemia initiating cells in acute myeloid and lymphoblastic leukemia (ALL). Most transplantation models have required conditioning in which the recipients were either irradiated or treated with chemotherapy prior to injection of human leukemia cells. In this study, we reported an undescribed model in which adult ALL cells were injected into unconditioned newborn nonobese diabetic severe combined immunodeficient mice via an intrahepatic route. MATERIALS AND METHODS Bone marrow (BM) and peripheral blood were collected from patients with ALL at diagnosis or relapse. CD34(+) selected lymphoblasts or mononuclear cells were transplanted as mentioned previously. Cells were also transplanted into sublethally irradiated adult mice via intravenous route for comparison. Leukemia engraftment was enumerated from mouse BM 6 to 18 weeks after transplantation. Clonality of the engrafting cells was examined based on IGH rearrangement and fluorescent in situ hybridization. RESULTS Five of 13 ALL samples engrafted into the recipient BM 6 to 18 weeks after transplantation. Engrafted cells recapitulated the immunophenotype and cytogenetic characteristics of the original samples. Engraftment in BM and peripheral blood was significantly correlated. Importantly, there was significant correlation of engraftment between this and the conventional adult nonobese diabetic severe combined immunodeficient mouse model involving irradiation. CONCLUSION Our results demonstrated that this unconditioned newborn mouse model could be used for enumeration of leukemia initiating cells in ALL and should be further evaluated.

Analysis of parameters that affect human hematopoietic cell outputs in mutant c-kit-immunodeficient mice

Xenograft models are transforming our understanding of the output capabilities of primitive human hematopoietic cells in vivo. However, many variables that affect posttransplantation reconstitution dynamics remain poorly understood. Here, we show that an equivalent level of human chimerism can be regenerated from human CD34+ cord blood cells transplanted intravenously either with or without additional radiation-inactivated cells into 2- to 6-month-old NOD-Rag1−/−-IL2Rγc−/− (NRG) mice given a more radioprotective conditioning regimen than is possible in conventionally used, repair-deficient NOD-Prkdcscid/scid-IL2Rγc−/−(NSG) hosts. Comparison of sublethally irradiated and non-irradiated NRG mice and W41/W41 derivatives showed superior chimerism in the W41-deficient recipients, with some differential effects on different lineage outputs. Consistently superior outputs were observed in female recipients regardless of their genotype, age, or pretransplantation conditioning, with greater differences apparent later after transplantation. These results define key parameters for optimizing the sensitivity and minimizing the intraexperimental variability of human hematopoietic xenografts generated in increasingly supportive immunodeficient host mice.

Differential NOD/SCID mouse engraftment of peripheral blood CD34+ cells and JAK2V617F clones from patients with myeloproliferative neoplasms

We evaluated the NOD/SCID engraftment of CD34(+) cells from polycythemia vera (PV) and secondary polycythemia patients (SP) and the JAK2V617F clone before and after transplantation. Peripheral blood CD34(+) cells were transplanted intra-femorally. In the injected BM, successful engraftment (>0.1%) occurred in 8/26 mice transplanted with CD34+ cells from 5/13 PV patients (median: 4.26%, range: 0.3-5.56%), in contrast to 0/14 mice from 9 SP patients (P=0.017). The engrafting PV cells were of multi-lineage. JAK2V617F/total JAK2 ratios decreased after transplantation (initial: 65.9% versus 6-week: 13.0%, P=0.001). Essential thrombocythemia (ET) BM cells also exhibited a similar decrease in JAK2V617F clone. The results suggested that events in addition to JAK2V617F are involved in the pathogenesis of PV and ET.

Sorafenib treatment of FLT3-ITD(+) acute myeloid leukemia

Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% of acute myeloid leukemias (AMLs) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD(+) AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21-84) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54-287) days but the FLT3 and downstream effectors remained suppressed. Gene expression profiling showed that leukemia cells that have become sorafenib resistant expressed several genes including ALDH1A1, JAK3, and MMP15, whose functions were unknown in AML. Nonobese diabetic/severe combined immunodeficiency mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain mutations at D835 were identified in leukemia initiating cells (LICs) from samples before sorafenib treatment. LICs bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggest that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations, and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors.