L. Bik To

Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia

We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.

The genomic landscape of hypodiploid acute lymphoblastic leukemia

The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole-genome and exome sequencing of 40 cases, identified two subtypes that differ in the severity of aneuploidy, transcriptional profiles and submicroscopic genetic alterations. Near-haploid ALL with 24–31 chromosomes harbor alterations targeting receptor tyrosine kinase signaling and Ras signaling (71%) and the lymphoid transcription factor gene IKZF3 (encoding AIOLOS; 13%). In contrast, low-hypodiploid ALL with 32–39 chromosomes are characterized by alterations in TP53 (91.2%) that are commonly present in nontumor cells, IKZF2 (encoding HELIOS; 53%) and RB1 (41%). Both near-haploid and low-hypodiploid leukemic cells show activation of Ras-signaling and phosphoinositide 3-kinase (PI3K)-signaling pathways and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.

Elevated Serum Levels of Stromal-Derived Factor-1α Are Associated with Increased Osteoclast Activity and Osteolytic Bone Disease in Multiple Myeloma Patients

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy able to mediate massive destruction of the axial and craniofacial skeleton. The aim of this study was to investigate the role of the potent chemokine, stromal-derived factor-1α (SDF-1α) in the recruitment of osteoclast precursors to the bone marrow. Our studies show that MM PC produce significant levels of SDF-1α protein and exhibit elevated plasma levels of SDF-1α when compared with normal, age-matched subjects. The level of SDF-1α positively correlated with the presence of multiple radiological bone lesions in individuals with MM, suggesting a potential role for SDF-1α in osteoclast precursor recruitment and activation. To examine this further, peripheral blood–derived CD14+ osteoclast precursors were cultured in an in vitro osteoclast-potentiating culture system in the presence of recombinant human SDF-1α. Although failing to stimulate an increase in TRAP+, multinucleated osteoclast formation, our studies show that SDF-1α mediated a dramatic increase in both the number and the size of the resorption lacunae formed. The increased osteoclast motility and activation in response to SDF-1α was associated with an increase in the expression of a number of osteoclast activation–related genes, including RANKL, RANK, TRAP, MMP-9, CA-II, and Cathepsin K. Importantly, the small-molecule CXCR4-specific inhibitor, 4F-Benzoyl-TE14011 (T140), effectively blocked osteoclast formation stimulated by the myeloma cell line, RPMI-8226. Based on these findings, we believe that the synthesis of high levels of SDF-1α by MM PC may serve to recruit osteoclast precursors to local sites within the bone marrow and enhance their motility and bone-resorbing activity. Therefore, we propose that inhibition of the CXCR4-SDF-1α axis may provide an effective means of treatment for MM-induced osteolysis.

Dasatinib Cellular Uptake and Efflux in Chronic Myeloid Leukemia Cells: Therapeutic Implications

Purpose: The organic cation transporter OCT-1 mediates active transport of imatinib. We recently showed that low OCT-1 activity is a major contributor to suboptimal response in chronic myeloid leukemia (CML) patients treated with imatinib. The relevance of OCT-1 activity and efflux pumps in determining intracellular uptake and retention (IUR) of dasatinib was assessed. Experimental Design: The effect of OCT inhibitors on [14C]dasatinib and [14C]imatinib IUR was compared using peripheral blood mononuclear cells from newly diagnosed CML patients. The role of efflux transporters was studied using ABCB1- and ABCG2-overexpressing cell lines and relevant inhibitors. Results: Unlike imatinib, there was no significant difference in the dasatinib IUR at 37°C and 4°C (P = 0.8), and OCT-1 inhibitors including prazosin did not reduce dasatinib IUR significantly. In CML mononuclear cells, prazosin inhibitable IUR was significantly higher for imatinib than dasatinib (6.38 versus 1.48 ng/200,000 cells; P = 0.002; n = 11). Patients with high OCT-1 activity based on their imatinib uptake had IC50dasatinib values equivalent to patients with low OCT-1 activity. Dasatinib IUR was significantly lower in ABCB1-overexpressing cell lines compared with parental cell lines (P < 0.05). PSC833 (ABCB1 inhibitor) significantly increased the dasatinib IUR (P < 0.05) and reduced IC50dasatinib (from 100 to 8 nmol/L) in K562-DOX cell line. The ABCG2 inhibitor Ko143 significantly increased dasatinib IUR in ABCG2-overexpressing cell lines and reduced IC50dasatinib. Conclusion: Unlike imatinib, dasatinib cellular uptake is not significantly affected by OCT-1 activity, so that expression and function of OCT-1 is unlikely to affect response to dasatinib. Dasatinib is a substrate of both efflux proteins, ABCB1 and ABCG2.

How I treat patients who mobilize hematopoietic stem cells poorly

Transplantation with 2-5 × 10(6) mobilized CD34(+)cells/kg body weight lowers transplantation costs and mortality. Mobilization is most commonly performed with recombinant human G-CSF with or without chemotherapy, but a proportion of patients/donors fail to mobilize sufficient cells. BM disease, prior treatment, and age are factors influencing mobilization, but genetics also contributes. Mobilization may fail because of the changes affecting the HSC/progenitor cell/BM niche integrity and chemotaxis. Poor mobilization affects patient outcome and increases resource use. Until recently increasing G-CSF dose and adding SCF have been used in poor mobilizers with limited success. However, plerixafor through its rapid direct blockage of the CXCR4/CXCL12 chemotaxis pathway and synergy with G-CSF and chemotherapy has become a new and important agent for mobilization. Its efficacy in upfront and failed mobilizers is well established. To maximize HSC harvest in poor mobilizers the clinician needs to optimize current mobilization protocols and to integrate novel agents such as plerixafor. These include when to mobilize in relation to chemotherapy, how to schedule and perform apheresis, how to identify poor mobilizers, and what are the criteria for preemptive and immediate salvage use of plerixafor.

Results of a phase 2 study of pacritinib (SB1518), a JAK2/JAK2(V617F) inhibitor, in patients with myelofibrosis

Pacritinib (SB1518) is a Janus kinase 2 (JAK2), JAK2(V617F), and Fms-like tyrosine kinase 3 inhibitor that does not inhibit JAK1. It demonstrated a favorable safety profile with promising efficacy in phase 1 studies in patients with primary and secondary myelofibrosis (MF). This multicenter phase 2 study further characterized the safety and efficacy of pacritinib in the treatment of patients with MF. Eligible patients had clinical splenomegaly poorly controlled with standard therapies or were newly diagnosed with intermediate- or high-risk Lille score. Patients with any degree of cytopenia were eligible. Thirty-five patients were enrolled. At entry, 40% had hemoglobin <10 g/dL and 43% had platelets <100 000× 10(9)/L. Up to week 24, 8 of 26 evaluable patients (31%) achieved a ≥35% decrease in spleen volume determined by magnetic resonance imaging and 14 of 33 (42%) attained a ≥50% reduction in spleen size by physical examination. Median MF symptom improvement was ≥50% for all symptoms except fatigue. Grade 1 or 2 diarrhea (69%) and nausea (49%) were the most common treatment-emergent adverse events. The study drug was discontinued in 9 patients (26%) due to adverse events (4 severe). Pacritinib is an active agent in patients with MF, offering a potential treatment option for patients with preexisting anemia and thrombocytopenia. This trial was registered at www.clinicaltrials.gov as #NCT00745550.

Measurement of In Vivo BCR-ABL Kinase Inhibition to Monitor Imatinib-Induced Target Blockade and Predict Response in Chronic Myeloid Leukemia

PURPOSE Intrinsic sensitivity to imatinib, based on measurement of inhibitory concentration 50% for imatinib, is variable in untreated patients with chronic myeloid leukemia (CML). This suggests that patient-tailored dosing may be more rational than a fixed dose for all. Dose optimization potentially could be based on accurate measurement of the level of BCR-ABL kinase inhibition achieved in vivo. PATIENTS AND METHODS In vivo kinase inhibition was measured by calculating the reduction in protein (p)--Crkl level in mononuclear blood cells taken from 49 CML patients at weekly intervals after imatinib therapy was commenced. RESULTS Greater than 50% inhibition (> 50% reduction in p-Crkl from baseline) was achieved by 21% of patients by days 7 to 14 (and maintained in all patients on days 21 to 28) and an additional 24% of patients achieved more than 50% inhibition by days 21 to 28. Thus, overall 45% of patients achieved more than 50% inhibition. All of these patients achieved major molecular responses by 24 months compared with 56% of the patients who failed to achieve 50% kinase inhibition (P < .001). Patients with less than 50% kinase inhibition were also more likely to have suboptimal responses. CONCLUSION In vivo BCR-ABL kinase inhibition can be assessed in the first month of imatinib therapy and may provide a valuable guide to optimization of dosage. The extent of BCR-ABL kinase inhibition is an excellent predictor of cytogenetic and molecular response. These observations suggest that dose adjustment based on in vivo measurements of drug-induced target inhibition could be applied in settings beyond imatinib and may be a more effective approach than using one dose for all patients in targeted anticancer therapy.

Targeted Disruption of the CXCL12/CXCR4 Axis Inhibits Osteolysis in a Murine Model of Myeloma-Associated Bone Loss†‡

The plasma cell (PC) malignancy, multiple myeloma (MM), is unique among hematological malignancies in its capacity to cause osteoclast (OC)‐mediated skeletal destruction. We have previously shown that elevated plasma levels of PC‐derived CXCL12 are associated with presence of X‐ray detectable osteolytic lesions in MM patients. To further investigate this relationship, plasma levels of CXCL12 and βCrossLaps, a marker of bone loss, were measured. A strong correlation between levels of CXCL12 and OC‐mediated bone resorption was identified. To confirm the OC‐activating potential of MM PC‐derived CXCL12 in vivo, we established a model of MM‐mediated focal osteolysis, wherein MM PC lines, such as RPMI‐8226, were injected into the tibias of nude mice. Implanting RPMI‐8226 gave rise to osteolytic lesions proximal to the tumor, resulting in a 5% decrease in bone volume (BV) compared with vehicle control. Importantly, bone loss was significantly inhibited with systemic administration of the CXCL12/CXCR4 antagonist T140. Furthermore, implanting CXCL12‐overexpressing RPMI‐8226 cells resulted in a 13% decrease in BV and was associated with increased OC recruitment proximal to the tumor, increased serum matrix metalloproteinase activity, and increased levels of collagen I degradation products. These findings confirm our hypothesis that MM PC‐derived CXCL12 stimulates the recruitment and activity of OC, thereby contributing to the formation of MM osteolytic lesions.

Therapeutic concentrations of dasatinib inhibit in vitro osteoclastogenesis

Dasatinib (BMS-354825, SPRYCEL; Bristol-Myers Squibb, New York, USA) is a second-generation ATP-competitive inhibitor of a subset of protein tyrosine kinases, including abl, Src-family kinases (c-Src, Lck, Hck, Yes, Fgr, Lyn and Fyn), and the plateletderived growth factor family members c-kit and platelet-derived growth factor-a and -b. Dasatinib displays clinical activity in chronic myeloid leukaemia patients who develop resistance to or are intolerant to the frontline chronic myeloid leukaemia chemotherapeutic imatinib mesylate (STI571, Gleevec; Novartis, Basel, Switzerland). Although designed to inhibit abl and/or Src protein kinases, tyrosine kinase inhibitors, like dasatinib and imatinib, show off-target effects. We have shown earlier that therapeutic concentrations of imatinib decrease osteoclast numbers and activity, at least in part through inhibition of the macrophage colony-stimulating factor (M-CSF) receptor, c-fms. Signalling through c-fms plays an essential role in the survival and activity of bone-resorbing osteoclasts, which act in conjunction with bone-forming osteoblasts to maintain skeletal health. In in vitro osteoclast cultures and animal models, such as op/op mice, an absence of signalling through c-fms results in an osteopetrotic phenotype because of deficient osteoclasts and osteoclast precursors. Recent evidence suggests that dasatinib, like imatinib, may potentially inhibit signal transduction through c-fms. In cultures of M-CSF-dependent Ba/F3 cells that ectopically expressed human c-fms, cell numbers were decreased by dasatinib treatment. In light of this observation, this study examined whether dasatinib could modulate osteoclast formation and/or activity, and whether any such effects were attributable to an inhibition of c-fms signal transduction. We examined the effect of dasatinib on osteoclast formation and activity in human and murine systems in vitro. Human CD14þ mononuclear cells (huCD14þ ) were isolated from the peripheral blood of healthy volunteers using Percoll gradient separation and a MACS negative selection monocyte kit (Miltenyi Biotech, Bergisch Gladbach, Germany). The cells were cultured with recombinant human (rh)M-CSF and rhRANKL, to induce osteoclastogenesis, supplemented with dasatinib or vehicle. After 14 days of culture, a significant decrease in the number of tartrate-resistant acid phosphatasepositive multinucleated cells was observed at 20 nM dasatinib (Po0.001; IC501⁄4 10.5 nM; Figure 1a). Murine osteoclastogenesis assays were established by explanting mouse bone marrow from the tibiae and femora of C57 BL/6 mice. Cells were incubated overnight at 37 1C in 5% CO2, to allow stromal cells to adhere, and non-adherent mouse bone marrow cells (mBM) were collected and used as monocyte/macrophage osteoclast precursor cells. In mBM cultures treated with rhM-CSF and rhRANKL for 6 days, osteoclast numbers were decreased at 10 nM dasatinib (Po0.01; Figure 1b) and osteoclast formation was completely abrogated at 20 nM dasatinib (IC501⁄4 8.0 nM; Figure 1b). Osteoclast size was also substantially reduced in cultures treated with 10 nM dasatinib compared with controls, in both human and murine systems (Figure 1c). The effect of dasatinib on osteoclast activity was also assessed in huCD14þ and mBM cultures established on calcium phosphate-coated slides. A significant inhibition of resorption was observed at X1.25 nM dasatinib in huCD14þ cultures (Po0.001; IC501⁄4 2.4 nM; Figures 1d and f), and at X2.5 nM dasatinib in mBM cultures (Po0.05; IC501⁄4 3.5 nM; Figures 1e and f), relative to vehicle controls. Thus, therapeutically relevant concentrations of dasatinib (Cmax1⁄4 110.0 nM) significantly reduced the formation and activity of osteoclasts from huCD14þ and mBM at IC50 concentrations of p10 nM dasatinib. As dasatinib may affect osteoclastogenesis through inhibition of c-fms, we investigated whether dasatinib could specifically inhibit c-fms kinase activity. Initially, the effect of dasatinib on c-fms-dependent cell proliferation was examined using FDC-P1 cells expressing c-fms protein (FDC-cfms). FDC-P1 cells are dependent on interleukin-3 for proliferation and are also rendered M-CSF-responsive by transfection with a c-fms expression construct. The resulting cells are dependent on either interleukin-3 or M-CSF for survival and proliferation. Treatment of rhM-CSF-stimulated FDC-cfms cells with dasatinib for 24 h significantly decreased cell numbers at concentrations of X50 nM (Po0.001; IC501⁄4 57.8 nM; Figure 2a). In contrast, the proliferation of FDC-P1 cells or FDC-cfms cells cultured in the presence of interleukin-3 (0.5 ng/ ml) was not significantly affected by dasatinib treatment at concentrations up to 100 nM (P1⁄4 0.9012 and P1⁄4 0.2008, respectively; Figure 2a). To determine whether dasatinib treatment could also inhibit the M-CSF-dependent proliferation/survival of primary bone marrow cells, the effect of dasatinib on mBM cells was assessed using a WST-1 assay (Tanaka, Madison, WI, USA). The relative number of viable, metabolically active cells per well, as detected by the quantification of mitochondrial dehydrogenase

Cotransplantation of placental mesenchymal stromal cells enhances single and double cord blood engraftment in nonobese diabetic/severe combined immune deficient mice.

Limited cell numbers in a unit restricts cord blood transplantation (CBT) in adults. We evaluated whether cotransplantation of placental mesenchymal stromal cells (MSCs) would enhance engraftment. Plastic adherent cells from placenta demonstrated typical characteristics of MSCs. In six individual experiments, 4 cohorts of 24 nonobese diabetic/severe combined immune deficient (NOD/SCID) mice were evaluated. Cohort 1 received 5 × 104 CD34+ cells from unit (U) one (SCBT); cohort 2 received 5 × 104 CD34+ cells from U1 + 4 × 104 MSCs (SCBT+MSCs); cohort 3 received 2.5 × 104 CD34+ cells from U1 + 2.5 × 104 CD34+ cells from U2 (double cord blood transplant [DCBT]); cohort 4 received 2.5 × 104 CD34+ cells from U1 + 2.5 × 104 CD34+ cells from U2 + 4 × 104 MSCs (DCBT+MSCs). Hematopoietic engraftment evaluated after 6 to 8 weeks, was similar in recipients of SCBT and DCBT. MSC cotransplantation demonstrated enhanced engraftment in DCBT (51.8 ± 6.8% versus 14.9 ± 6.5%; p = .04) with an increased trend in SCBT (48.7 ± 7.7% versus 17.5 ± 6.1%; p = .07). In DCBT, cotransplantation of placental MSCs reduced single cord dominance. Self‐renewal capacity was assessed by serial transplantation in secondary recipients infused with engrafted human cells from primary mice transplanted with or without MSCs. In secondary transplant experiments, 13 of 17 evaluable mice engrafted at levels of 1% to 6.5%. Despite enhanced engraftment in primary mice, long‐term engraftment capacity was unaltered with MSC cotransplantation. Imaging studies showed MSCs migrated to pelvic region and improved cord blood (CB) CD34+ homing. Cotransplantation of placental MSCs enhanced cord blood engraftment and may act by improving homing of CD34+ cells. STEM CELLS 2009;27:2293–2300