Chronic Myeloid Leukemia Blast Crisis Research Articles

Jak2 Regulates Bcr-Abl In CD34+ Cells From Imatinib Mesylate-Resistant CML Patients.

AbstractAbstract 1220Despite the success of imatinib mesylate (IM), complete molecular remission is difficult to achieve in IM-treated CML patients. After continuing treatment with IM, a population of leukemic stem/progenitor cells remains, which are insensitive to IM. Our previous studies have shown that Jak2 regulates Bcr-Abl signaling and maintains levels of Bcr-Abl in CML cell lines, fresh cells from blast crisis patients, and mouse hematopoietic cells transformed by wild-type and imatinib-resistant mutants of BCR-ABL (Samanta et al. ASH 2009 meeting, submitted). We hypothesize that Jak2 plays similar role in Bcr-Abl+ CD34+ stem/progenitor cells, and that inhibition of Jak2 will induce apoptosis by inhibiting critical signaling pathways. In our current studies we have examined Bcr-Abl CD34+ progenitor cells from CML blast crisis patients. These cells are resistant to IM but quite sensitive to cell death induction by the selective Jak2 inhibitor TG101209 at 5–10 μM concentrations for 48 h. Similar results were obtained with cells from IM-resistant accelerated phase and IM-resistant chronic phase patients. Western blotting of CD34+ cells from an IM-resistant blast crisis CML patient established that a four h treatment with TG101209 at 10 μM severely reduced levels of Bcr-Abl protein and levels of phosphotyrosine 177 within Bcr-Abl.To begin to assess whether Jak2 effects on Bcr-Abl signaling also occur in early stage Bcr-Abl induced disease, we assayed CD34+ cord blood cells transformed by Bcr-Abl in short-term culture (Chu S et al. Cancer Res 2007; 67: 7045–7053). The results showed that a six h treatment with TG101209 (10 μM) reduced levels of: Jak2 protein; activated Jak2 (lower levels of pTyr 1007/8); Tyr 177 within Bcr-Abl; and the Bcr-Abl protein itself. These results support our earlier findings that Jak2 regulates Bcr-Abl signaling in CML cell lines (Samanta AK, et al. Cancer Res 66 (13):6468-72, 7/2006; Samanta AK, et al., Oncogene 28 (14):1669-81, 4/2009; Tao W., et al., Oncogene 27(22):3194-200, 5/2008). Furthermore, these results suggest that Jak2 plays a critical role in progenitor CML cells from early stage and late stage patients with CML disease. Disclosures:Bhatia:Novartis: Consultancy.

Targeting the Leukemia-Associated Hypoxic Microenvironment with Hypoxia-Activated Prodrug PR-104

AbstractAbstract 868Interactions between leukemia cells and the bone marrow (BM) microenvironment are known to promote leukemia cell survival and confer resistance to drugs commonly used in the management of this disease. We investigated the protective role of hypoxia in the BM microenvironment. We observed a marked expansion of hypoxic niches in the bone marrow of immunodeficient mice engrafted with the acute lymphoblastic leukemia (ALL) cell line Nalm6 and with primary ALL cells, as detected by the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. We further demonstrated induction of the chemokine receptor CXCR4, and of the enzyme carbonic anhydrase 9 (CAIX), both targets of Hypoxia-Inducible Factor 1α (HIF-1a), in hypoxic areas of BM from mice harboring the Nalm6 xenografts. Furthermore, we used a CML blast crisis model to evaluate the time course of hypoxia expansion in the BM. In C57Bl6/J mice engrafted with murine HSCs co-expressing BCR/ABL and Nup98 we observed a time-dependent increase in PIM positive areas which coincided with the presence of GFP positive cells. In line with these findings, HIF-1α was highly expressed in BM biopsies from newly diagnosed ALL patients (n=15) but was significantly reduced when the patients achieved complete remission (CR). Culture under hypoxic conditions (1% O2) conferred resistance of pre-B ALL cells REH, Nalm-6, and of AML OCI-AML3 leukemic cells against several chemotherapeutic agents including vincristine, methotrexate and idarubicin (% of Annexin V(+) cells at 21% vs 1%O2; REH plus 1ng/ml vincristine:88.5+/−2.3 vs 18.1+/−10.3; REH plus 0.25uM etoposide: 86.4 +/−7 vs 14.4+/−8.6; Nalm6 plus 10ng/ml methotrexate: 33.4+/−2.7 vs 4.6+/−1; OCI-AML3 plus 50ng/ml idarubicin: 28.45+/−5 vs 12.76+/−1) . Taken together, these results provide rationale for examining the potential of hypoxia-activated pro-drugs to eliminate leukemia progenitor cells within hypoxic niches. To this end, we tested the hypoxia-activated prodrug PR104, a dinitrobenzamide nitrogen mustard that is reduced to its active metabolites under hypoxic conditions (Patterson et al., Clin Can Res 2007). In vitro, PR-104 induced cell death in three different leukemia cell lines (Nalm6, REH and the AML line Molm13) selectively under hypoxic (pO2 1%) conditions. The anti-leukemic efficacy of PR-104 as a single agent was next examined in several in vivo leukemia models. Administration of PR-104 prolonged survival and decreased leukemia burden of 1) NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML; 2) NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL; and 3) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice injected with Nalm6-luciferase ALL cells. Next, we evaluated anti-tumor effects of PR-104 at clinically relevant doses (200, 100 and 50 mg/kg) and at maximal tolerated dose (550 mg/kg) in two ALL xenograft models (a T-lineage ALL and a B-cell precursor ALL) (Figure 1, studies supported by NCI NO1CM42216 and by PPTP contract NO1-CM91001-03). Compared to vehicle control, PR-104 significantly delayed progression of the T-ALL xenografts at all doses tested and at three of the four doses (550, 200 and 100 mg/kg) of B-cell pre-ALL, which resulted in significantly increased event-free survival (EFS) of mice in the treatment groups.Altogether, these findings strongly suggest that targeting hypoxia is feasible. If successful, this approach may significantly impact leukemia therapy and ultimately improve patient survival. This concept is currently being tested in an ongoing Phase I clinical trial of PR-104 in relapsed/refractory AML patients. [Display omitted] Disclosures:Wilson:PROACTA: Equity Ownership. Konopleva:PROACTA: PI on clin trial funded by Proacta.

SIRT1 Inhibition Induces Apoptosis In Human CML Progenitors by Enhancing p53 Acetylation and Activation

AbstractAbstract 200Imatinib mesylate (IM) treatment is effective in inhibiting CML primitive progenitor growth but induces only modest levels of apoptosis. Improved approaches to enhance elimination of residual CML progenitors in IM-treated patients are required. The NAD+ dependent deacetylase SIRT1 is a stress-response gene that is expressed at higher levels in CML compared to normal CD34+ progenitors. We have shown that inhibition of SIRT1 expression using lentivirus-mediated SIRT1 shRNA expression results in modest induction of apoptosis in CML progenitors and significantly enhanced apoptosis in combination with IM (Blood 2009, 114: 189). SIRT1 inhibition does not induce apoptosis in normal progenitors or increase their sensitivity to IM. SIRT1 can potentially regulate the acetylation of several transcription factors, including the p53 tumor suppressor protein. In contrast to several other cancers, p53 mutations are rare in CP CML, suggesting that p53 may still be subject to activation in CML progenitors. However we have observed that p53 levels are reduced in IM-treated CML CD34+ progenitors. We were therefore interested in investigating whether increased apoptosis of CML progenitors following SIRT1 inhibition was related to enhancement of p53 activity via protein acetylation. We observed that inhibition of SIRT1 using shRNA resulted in increased acetylation of p53 in CML CD34+ cells without increase in total p53 expression on both western blotting and flow cytometry. SIRT1 inhibition also increased p53 acetylation in IM-treated cells. Acetylated p53 was observed to localize to the nuclei of CML CD34+ cells on immunofluorescence microscopy. Q-PCR analysis revealed increased expression of the p53 transcriptional targets, GFI-1 and Necdin, in SIRT1 knockdown CML CD34+ cells (Necdin, Si versus Ctrl, 2.7±0.4 fold, p<0.05, n=3; GFI-1, Si versus Ctrl, 2.4±0.4 fold, p<0.05, n=3). These results suggest that SIRT1 inhibition results in increased p53 acetylation, nuclear localization and transcriptional activity in CML CD34+ cells. To further investigate the role of p53 in mediating the effects of SIRT1 inhibition we concomitantly knocked down both p53 and SIRT1 in CML CD34+ cells. Inhibition of p53 expression by lentivirus mediated delivery of p53 shRNA significantly enhanced growth and reduced apoptosis of SIRT1 knockdown CML CD34+ cells (14±2% apoptosis with SIRT1 knockdown, 7±2% apoptosis with combined SIRT1 and p53 knockdown, p<0.05, n=3). These results confirm an important role for p53 in SIRT1 mediated effects in CML progenitors. SIRT1 inhibition did not inhibit growth or induce apoptosis in CML blast crisis K562 cells, which are p53 null. To further determine the specific role of p53 acetylation in mediating SIRT1 effects, we expressed both wild type and acetylation-deficient p53 constructs in K562 cells. K562 cells ectopically expressing the wild type p53 gene demonstrated significant growth inhibition and apoptosis following SIRT1 knockdown (SIRT1 shRNA, 18±5% versus Ctrl shRNA, 8±3%, p<0.05), increased levels of acetylated p53, and enhanced transactivation of a p53 reporter containing the mdm2 promoter cloned upstream of the luciferase gene (p<0.05). In contrast, K562 cells transfected with an acetylation-defective p53 gene (with all eight acetylation sites mutated) did not demonstrate significant growth inhibition or apoptosis following SIRT1 inhibition. These results indicate that the inhibitory effect of SIRT1 on CML cells is dependent on p53 acetylation. We conclude that inhibition of SIRT1 enhances p53 acetylation and transcriptional activity resulting in enhanced apoptosis of CML progenitors. SIRT1 is a potentially druggable target, and several groups are actively developing SIRT1 inhibitory compounds. Activation of p53 via SIRT1 inhibition represents an attractive approach to eradicate CML stem cells in combination with IM or other treatments. Disclosures:No relevant conflicts of interest to declare.

A New BCR-ABL1 Mutation (L248R) Is Highly Resistant to Imatinib, Bosutinib, Nilotinib and Dasatinib, but Can Be Inhibited by AP-24534 and DCC-2036.

AbstractAbstract 3398Chronic Myeloid Leukemia (CML) treatment was radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion kinase Bcr-Abl. Second generation ATP-competitive tyrosine kinase inhibitors (TKIs) bosutinib (BOS), nilotinib (NIL) and dasatinib (DAS) further improved CML therapy. However, resistance to TKIs occurs in a variable proportion of patients; it can arise from different mechanisms but in the majority of cases it is due to Bcr-Abl point mutations that alter directly or indirectly the drug-protein binding. Over 70 mutations have been described in patients, with T315I showing resistance to IM, NIL, BOS and DAS. Here we report the discovery of a new P-loop mutant (L248R) that is highly resistant to all currently available inhibitors. L248R was identified in a lymphoid Blast Crisis CML patient. The patient initially presented with an IM-resistant F359I mutation. A cytogenetic and molecular response was obtained with BOS, but after 1 year haematological relapse developed; at this point a F359I/L248R clone was identified. L248R was previously reported only in an in vitro during a mutagenesis screen involving IM (Bradeen et al. 2006) and in a study with the T315I inhibitor SGX393 (O'Hare et al. 2008). L248R was never isolated from a clinical sample. Activity profile of BOS, IM, DAS and NIL against L248R, F359I and F359I/L248R was performed (Table). Stable transfectant Ba/F3 cells were generated and the TKIs anti-proliferative activity was determined. The relative IC50 increase over wild type Bcr-Abl (Relative Resistance, RR) was calculated. We classified RR values in four categories: sensitive (RR≤ 2), moderately resistant (RR between 2.1 and 4), resistant (RR between 4.1 and 10) or highly resistant (RR>10). In all cases a RR >10 was obtained, for L248R. Recently new compounds were developed to overcome resistance generated by the T315I mutant. Among them AP-24534 (AP), a panBcr-Abl inhibitor (O'Hare et al. 2009) and the switch pocket inhibitor DCC-2036 (DCC) were reported as potently active against the T315I mutation. Both compounds showed activity against L248R (RR 6.2 and 0.4), although the activity against the double mutant F359I/L248R was reduced especially for AP (RR 17.7 and 1.0). The activity profile of AP and DCC against L248R and a panel of 26 mutated forms of Bcr/Abl covering the most common mutations is also presented (Table). Activity of BOS, IM, DAS and NIL is also reported for comparison. According to our data, only mutation E255V is classified as highly resistant to AP, in addition to F359I/L248R: 6/26 mutations are considered “resistant” to AP (L248R, G250E, G252H, E255K, F359V, H396R) and 3/26 to DCC (D276G, E279K, F317V). The huge difference in the IC50 values for AP between Bcr/Abl wild type (2.1 nM) and parental Ba/F3 cells (>1000 nM) could render some of the mutants with high RR values to AP still sensitive to this inhibitor. It is also important to note that, according to our data, every mutation analysed shows sensitivity to at least one of the tested TKIs. In conclusion we describe a novel mutation that is highly resistant to the commonly used Bcr-Abl TKIs but which is inhibited by AP and DCC. Modelling data on the L248R mutant in complex with different TKIs will also be presented [Display omitted] IC 50 values are based on tritiated thymidine incorporation assay. Results are an average of at least 3 independent experiments Disclosures:Wise:Deciphera Pharmaceuticals: Employment. Flynn:Deciphera Pharmaceuticals: Employment. Gambacorti-Passerini:Pfizer pharmaceuticals: Research Funding.

Microenvironmental Changes In An In Vivo Model of Myeloid Leukemia Negatively Regulate Osteoblastic Cells.

AbstractAbstract 1219Patients with myelogenous leukemias can present with symptoms of bone pain and pathologic fractures, however little is known about the interactions between malignant cells and the bone marrow microenvironment. Additionally leukemia is known to severely interfere with normal hematopoiesis. To further characterize interactions between leukemic cells and the microenvironment, we used a model of blast crisis CML (bcCML) in which immature murine hematopoietic cells are engineered to express the BCR/ABL and Nup98/HoxA9 translocation products. Injection of these cells into naïve mice results in rapid accumulation of leukemic cells in the bone marrow (Dash et al. PNAS, 2002). We investigated the effect of leukemia on the bone marrow microenvironment by first performing immunohistochemical analyses. Leukemic cells were observed to preferentially localize in close contact with bony trabeculae. In addition, leukemic mice also exhibited a dramatic loss of trabecular bone as measured by micro-CT scanning (22.8 ± 1.5% vs 13.6 ± 1.5%, BV/TV n=5 in each group p=0.0048), prompting us to examine bone resorption and the abundance of osteoclasts. Histologic sections from leukemic mice showed an increase in mature osteoclasts (TRAP+, multinucleated cells) at the endosteal surface of the long bones (51 ± 4 OC/section vs 64 ± 3 OC/section p=0.0229). Additionally leukemic mice had a 50% increase in serum C-telopeptide (CTX), a well-established marker of global bone resorption (15.5 ± 0.3 ng/ml vs 21.8 ± 1.0 ng/ml, p=0.0003). Therefore, the presence of leukemic cells appears to strongly stimulate osteoclastogenesis and bone resorption. In addition to increased osteoclasts, a rapid and severe reduction in bone formation was identified in leukemic mice by decreased serum osteocalcin, a well-established marker of bone formation (70.8 ± 6.9 ng/ml vs 39.9 ± 3.2 ng/ml, p=0.0036). To determine effects of leukemia on bone-forming cells, we analyzed osteoblastic cells from the long bones of leukemic animals. Marrow was flushed from the bone and minced bone fragments were digested in collagenase. Cells isolated in this fashion have strong osteoblastic activity and can support hematopoietic stem cells (HSCs) (Chitteti et al, Blood 2010). Leukemic cells were present in the isolated fraction confirming that leukemic cells were closely associated with the bone and not entirely removed when the marrow was flushed. Total cells isolated by collagenase digestion from the long bones of leukemic mice were cultured with leukemic cells and evaluated for osteoblastic colony-forming ability. These cultures demonstrated a reduced ability to form osteoblastic colonies compared to controls (26 ± 2 colonies vs 13 ± 2 colonies p=0.0014). Freshly isolated cells were CD45 depleted to remove leukemic cells and again evaluated for colony-forming ability. CD45 negative cells from leukemic mice also demonstrated reduced ability to form mineralizing osteoblastic colonies in vitro compared to controls when an identical number of cells were cultured (10 ± 2 colonies vs 0 colonies) suggesting that their previous exposure to leukemic cells in vivo was sufficient to decrease their osteoblastic activity. Further in vivo osteoblastic evaluation in leukemic mice showed reduced immunohistochemical staining for the osteoblastic marker osteopontin at the endosteal surface, supporting a leukemic-induced reduction of the mature osteoblastic population. These data demonstrate a severe reduction in both number and function of osteoblastic cells in a leukemic environment. Together these data show severe effects of leukemia on both osteoblastic and osteoclastic cells, which could contribute to the bone-specific problems associated with leukemic disease. Moreover, we propose that osteoblastic defects observed in this model may contribute to the leukemia-induced inhibitory effects on normal hematopoiesis. Studies are ongoing to assess the microenvironmental support for normal hematopoiesis in the leukemic setting and to identify the leukemic signals that modify the bone marrow microenvironment. Disclosures:No relevant conflicts of interest to declare.

A New Remedy for Blast-Crisis CML?

A New Remedy for Blast-Crisis CML?

P73. Regulation of myeloid leukemia by the cell fate determinant Musashi

Chronic myelogenous leukemia (CML) can progress from an indolent chronic phase to an aggressive blast crisis phase but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML to show that disease progression is regulated by the Musashi-Numb signaling axis. Specifically we find that chronic phase is marked by high and blast crisis phase by low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced phase disease in vivo. As a possible explanation for the decreased levels of Numb in blast crisis phase, we show that NUP98-HOXA9, an oncogene associated with blast crisis CML, can trigger expression of Musashi 2 (Msi2) an RNA binding protein which in turn represses Numb. Importantly, loss of Msi2 significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally, we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi-Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for therapy of aggressive leukemias.

Functional p53 is required for effective execution of telomerase inhibition in BCR-ABL–positive CML cells

In chronic myeloid leukemia (CML), increased cellular turnover of hematopoietic cells driven by the oncogene BCR-ABL leads to accelerated telomere shortening despite increased telomerase activity. It has been postulated that shortened telomeres, particularly in the context of increased telomerase activity, might facilitate accumulation of genetic aberrations and, consequently, disease progression from chronic phase to accelerated phase and blast crisis. Therefore, inhibition of telomerase might be a promising approach in CML therapy. To investigate the therapeutic potential of telomerase inhibition in this model disorder, we used a small molecule telomerase inhibitor, BIBR1532 as well as expression of a dominant-negative mutant of hTERT (DNhTERT-IRES-GFP) in the p53-negative CML blast crisis cell line K562 and characterized the effects in long-term culture. Furthermore, we expressed an inducible p53 construct (vector pBabe-p53ER(tam)) via retroviral transduction in cells with critically short telomeres and in cells with a normal telomere length to explain the role of the tumor suppressor in response to critical telomere shortening in BCR-ABL-positive cells. BIBR1532-treated bulk cultures did not show altered growth kinetics despite significant telomere shortening to a critical length of approximately 5 kb. In comparison, DNhTERT-expressing clones either lost telomere length, leading to a significant but transient slow down in proliferation but eventually all escaped senescence/crisis (group I) or, alternatively, remained virtually unaffected despite measurable telomerase inhibition (group II). Further analyses of group I clones revealed impaired DNA damage response and an accumulation of dicentric chromosomes. However, upon restoration of p53 in telomerase-negative K562 clones with critically short telomeres, immediate reinduction of apoptosis and complete eradication of cells was observed, whereas vector control cells continued to escape from crisis. These results suggest that the success of strategies aimed at telomerase inhibition in CML is highly dependent on the presence of functional p53 and should be explored preferentially in chronic phase CML.

Activation-Induced Cytidine Deaminase Accelerates Clonal Evolution in BCR-ABL1–Driven B-Cell Lineage Acute Lymphoblastic Leukemia

Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and immunoglobulin (Ig) class switch recombination in germinal center (GC) B cells. Occasionally, AID can target non-Ig genes and thereby promote GC B-cell lymphomagenesis. We recently showed that the oncogenic BCR-ABL1 kinase induces aberrant expression of AID in pre-B acute lymphoblastic leukemia (ALL) and lymphoid chronic myelogenous leukemia blast crisis. To elucidate the biological significance of aberrant AID expression, we studied loss of AID function in a murine model of BCR-ABL1 ALL. Mice transplanted with BCR-ABL1-transduced AID(-/-) bone marrow had prolonged survival compared with mice transplanted with leukemia cells generated from AID(+/+) bone marrow. Consistent with a causative role of AID in genetic instability, AID(-/-) leukemia had a lower frequency of amplifications and deletions and a lower frequency of mutations in non-Ig genes, including Pax5 and Rhoh compared with AID(+/+) leukemias. AID(-/-) and AID(+/+) ALL cells showed a markedly distinct gene expression pattern, and AID(-/-) ALL cells failed to downregulate a number of tumor-suppressor genes including Rhoh, Cdkn1a (p21), and Blnk (SLP65). We conclude that AID accelerates clonal evolution in BCR-ABL1 ALL by enhancing genetic instability and aberrant somatic hypermutation, and by negative regulation of tumor-suppressor genes.

HLA-C Antigen Mismatch Is Associated with Worse Outcome in Unrelated Donor Peripheral Blood Stem Cell Transplantation

The association between HLA matching and outcome in unrelated-donor peripheral blood stem cell (PBSC) transplantation has not yet been established. In the present study, a total of 1933 unrelated donor-recipient pairs who underwent PBSC transplantation between 1999 and 2006 for acute myelogenous leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, or chronic myelogenous leukemia and received high-resolution HLA typing for HLA-A, -B, -C, -DRB1, -DQA1, and -DQB1 were included in the analysis. Outcomes were compared between HLA-matched and HLA-mismatched pairs, adjusting for patient and transplant characteristics. Matching for HLA-A, -B, -C, and -DRB1 alleles (8/8 match) was associated with better survival at 1 year compared with 7/8 HLA-matched pairs (56% vs 47%). Using 8/8 HLA-matched patients as the baseline (n = 1243), HLA-C antigen mismatches (n = 189) were statistically significantly associated with lower leukemia-free survival (relative risk [RR], 1.36; 95% confidence interval [CI], 1.13-1.64; P = .0010), and increased risk for mortality (RR, 1.41; 95% CI, 1.16-1.70; P = .0005), treatment-related mortality (RR, 1.61; 95% CI, 1.25-2.08; P = .0002), and grade III-IV graft-versus-host disease (RR, 1.98; 95% CI, 1.50-2.62; P < .0001). HLA-B antigen or allele mismatching was associated with an increased risk for acute GVHD grade III-IV. No statistically significant differences in outcome were observed for HLA-C allele (n = 61), HLA-A antigen/allele (n = 136), HLA-DRB1 allele (n = 39), or HLA-DQ antigen/allele (n = 114) mismatches compared with 8/8 HLA-matched pairs. HLA mismatch was not associated with relapse or chronic GVHD. HLA-C antigen-mismatched unrelated PBSC donors were associated with worse outcomes compared with 8/8 HLA-matched donors. The study's limited power due to small sample size precludesconclusions about other mismatches.

Expression of Slug Is Regulated by c-Myb and Is Required for Invasion and Bone Marrow Homing of Cancer Cells of Different Origin

In metastatic cancer cells, the process of invasion is regulated by several transcription factors that induce changes required for migration and resistance to apoptosis. Slug (SNAI2, Snail2) is involved in epithelial mesenchymal transition in physiological and in pathological contexts. We show here that in embryonic kidney, colon carcinoma, chronic myeloid leukemia-blast crisis, and in neuroblastoma cells, expression of Slug is transcriptionally regulated by c-Myb via Myb binding sites in the 5'-flanking region and in the first intron of the slug gene. In embryonic kidney and neuroblastoma cells, c-Myb induced vimentin, fibronectin, and N-cadherin expression and membrane ruffling via actin polymerization consistent with the acquisition of a mesenchymal-like phenotype. Furthermore, down-regulation of endogenous c-Myb levels in colon carcinoma cells led to increased expression of E-cadherin and reduced levels of vimentin. Some of these changes are predominantly Slug-dependent as Slug silencing via RNA interference (RNAi) reverts the cells to a quasi-parental condition. Changes in gene expression and morphology induced by c-Myb-activated Slug correlated with increased ability to migrate (embryonic kidney) and to invade through a Matrigel membrane (embryonic kidney, colon carcinoma, neuroblastoma). c-Myb-dependent Slug expression was also essential for the homing of chronic myeloid leukemia K562 cells to the bone marrow. In summary, we show here that the proto-oncogene c-Myb controls Slug transcription in tumor cells of different origin. Such a regulatory pathway contributes to the acquisition of invasive properties that are important for the metastatic process.

Leukemia Lineage-Specific Chimerism Analysis and Molecular Monitoring Improve Outcome of Donor Lymphocyte Infusions

A retrospective analysis was performed of 118 patients with hematologic malignancies who received donor lymphocyte infusions (DLIs) after allogeneic stem cell transplantation (ASCT). Treatment was either given because of hematologic relapse (n = 44), molecular/cytogenetic relapse (n = 52), or other causes (n = 22). Molecular relapse was in most cases based on leukemia lineage-specific chimerism analysis. Patients with acute leukemia and myelodysplastic syndrome showed a 3-year survival of 42% if DLI treatment was given because ofmolecular relapse, compared to 16% in hematologic relapse (P < .006). In multivariate analysis, there was a correlation between response to DLI and nonhematologic relapse (risk ratio [RR] 3.36, P = .001), chronic graft-versus-host disease (cGVHD) (RR = 1.51, P = .005), and late relapse (RR = 2.06, P = .017). The overall incidences of acute GVHD (aGVHD) grades I-II and grades III-IV aGVHD were 33% and 8.5%, respectively. Probability of cGVHD was 33%. The development of aGVHD or cGVHD did not significantly influence the response of DLI in patients with molecular/cytogenetic relapse. However, the development of cGVHD was significantly associated with a better response in patients with hematologic relapse because only 4 of 29 patients without cGVHD responded compared to 7 of 12 with cGVHD (P = .007). The development of cGVHD increased significantly if DLI was given >12 months after ASCT (46% versus 27%, P = .04). In contrast, time between ASCT and start of DLI treatment had no significant influence on the risk of developing aGVHD. To conclude, monitoring of leukemia lineage-specific chimerism is of utmost importance for DLI response after ASCT.

Long-term outcomes in patients with high-risk myeloid malignancies following matched related donor hematopoietic cell transplantation with myeloablative conditioning of BU, etoposide and CY

Patients with high-risk or advanced myeloid malignancies have limited effective treatment options. These include high-dose therapy followed by allogeneic hematopoietic cell transplantation (HCT). We report a single-institution, long-term follow-up of 96 patients, median age 50 (range, 20-60) years, who received HLA-matched related HCT between 1992 and 2007. All patients were treated with a uniform preparatory regimen intended to enhance the widely used regimen of BU and CY that included: BU 16.0 mg/kg (days -8 to -5), etoposide 60 mg/kg (day -4), CY 60 mg/kg (day -2) with GVHD prophylaxis of CsA or FK506 and prednisone. Disease status at transplantation was high-risk AML (n=41), CML in second chronic phase or blast crisis (n=8), myelofibrosis and myeloproliferative disorders (n=8), and myelodysplasia (n=39). Thirty-six percent (n=35) of patients received BM whereas 64% (n=61) received G-CSF-mobilized PBPC. With a median follow-up of 5.6 years (range, 1.6-14.6 years) actuarial 5-year OS was 32% (95% CI 22-42) and 5-year EFS was 31% (95% CI 21-41). Relapse rate was 24% (95% CI 15-33) at 2 and 5 years. Nonrelapse mortality was 29% (95% CI 20-38) at day 100 and 38% (95% CI 29-47) at 1 year. Cumulative incidence of acute (grade II-IV) and extensive chronic GVHD was 27% (95% CI 18-36) and 29% (95% CI 18-40), respectively. There was no statistically significant difference in OS (31 vs 32%, P=0.89) or relapse rates (17 vs 28%, P=0.22) for recipients of BM vs PBPC, respectively. These results confirm that patients with high-risk or advanced myeloid malignancies can achieve long-term survival following myeloablative allogeneic HCT with aggressive conditioning.

Use of nilotinib to induce responses with 24-month (mo) minimum follow-up in patients (pts) with chronic myeloid leukemia in blast crisis (CML-BC) resistant to or intolerant of imatinib.

6510 Background: Nilotinib is a highly potent and selective BCR-ABL inhibitor with efficacy in pts with imatinib-resistant and -intolerant CML in chronic phase (CP) and accelerated phase (AP). Results from pts with CML-BC treated with nilotinib in the phase II 2101 trial are presented. Methods: Pts were treated with nilotinib 400 mg bid. Primary endpoint was confirmed hematologic response (HR). Results: 136 pts with imatinib-resistant (82%) or -intolerant (18%) CML-BC were treated with nilotinib and followed for a minimum of 24 mo. 105 pts had myeloid blast crisis (MBC) and 31 pts had lymphoid blast crisis (LBC). Baseline BCR-ABL mutations were detected in 30% of pts with MBC and 61% with LBC; T315I mutations were detected in 1% of pts with MBC and 7% with LBC. Clonal evolution was detected in 54% of all pts. Best prior response to imatinib was stable or progressive disease in 46%, with only 10% achieving prior complete HR (CHR) and 13% major cytogenetic response (MCyR). Nilotinib resulted in HR in 24% of pts with MBC and 19% with LBC. CHR occurred in 13% of pts with MBC and LBC. Responses were rapid and durable. For pts with MBC, median time to HR was 1 mo, and median duration of HR was 26 mo (range, 1.25-29.08); 60% of pts maintained HR at 24 mo. Median duration of HR was 3.6 mo for pts with LBC and no pt maintained HR at 24 mo. Nilotinib induced MCyR and CCyR in 38% and 30% of pts with MBC and 52% and 32% with LBC, respectively. In MBC pts, median time to MCyR was 1.8 mo, and median duration of MCyR was 11 mo (range, 0.03-29.08); 44% of pts maintained MCyR at 24 mo. Median duration of MCyR was 3.2 mo for pts with LBC and no pt maintained MCyR at 24 mo. Survival was 42% at 12 mo and 27% at 24 mo; 12 pts with MBC and 2 pts with LBC had transplants following nilotinib therapy. Nonhematologic adverse events were generally mild and manageable and similar to those observed in pts with CML-CP. Conclusions: Nilotinib is effective therapy in pts with imatinib-resistant or -intolerant CML-BC, generating rapid and durable responses for pts with MBC. Nilotinib exhibits a favorable safety profile in CML-BC patients. No new safety concerns were observed with 24 mo minimum follow-up. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Bristol-Myers Squibb, Novartis Novartis Bristol-Myers Squibb, Novartis Bristol-Myers Squibb, Genzyme, Novartis

BCR–ABL: a multi-faceted promoter of DNA mutation in chronic myelogeneous leukemia

The role of the BCR-ABL oncogene in the progression of chronic myeloid leukemia (CML) to blast crisis (BC) is unknown. The appearance of chromosomal aberrations in patients with CML BC has led to many attempts to elucidate a mechanism whereby BCR-ABL affects DNA damage and repair. BCR-ABL-expressing cells have been found to accumulate genetic abnormalities, but the mechanism leading to this genomic instability is controversial. In this study, we review the effects of BCR-ABL on DNA repair mechanisms, centrosomes, checkpoint activation and apoptosis. BCR-ABL has diverse effects on these mechanisms, but which of these effects are necessary for the progression of CML to BC is still unresolved.

Effective Targeting of Quiescent Chronic Myelogenous Leukemia Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate

Imatinib mesylate (IM) induces remission in chronic myelogenous leukemia (CML) patients but does not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. Here we investigated the ability of HDAC inhibitors (HDACis) to target CML stem cells. Treatment with HDACis combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone, and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACis with IM markedly diminished LSCs in a transgenic mouse model of CML. The interaction of IM and HDACis inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents an effective strategy to target LSCs in CML patients receiving tyrosine kinase inhibitors.

Methylation patterns in CD34 positive chronic myeloid leukemia blast crisis cells

Treatment results of chronic myeloid leukemia blast crisis are unsatisfactory, even with second generation tyrosine kinase inhibitors and allogeneic stem cell transplantation. Loss of tumor suppressor gene activity is one of the events that is associated with progression to blast crisis. Next to

Abstract LB-258: Smoothening the way for human leukemia stem cell inhibition in chronic myeloid leukemia

Abstract Chronic myeloid leukemia (CML) progresses from a chronic phase (CP) state that harbors the BCR-ABL fusion gene, to CML blast crisis (BC), characterized by activation of -catenin within granulocyte-macrophage progenitors (GMP) (Jamieson et al, NEJM 2004). BC CML GMP serially transplant leukemia in vivo and form myeloid sarcomas (Abrahamsson et al, PNAS 2009). Wnt, GSK3 and Shh family molecules are implicated in driving leukemia progression and because GSK3 is a negative regulator of both Wnt and Shh signaling pathways, we evaluated whether GSK3 deregulation activates -catenin and Gli simultaneously. We investigated whether treatment with a SMO antagonist in combination with dasatinib decreases human BC CML engraftment in vivo. BC CML tumor cells were treated for 0 and 7 days in vitro with vehicle (DMSO) or SMO antagonist (Pfizer; PF-04449913) and harvested for Q-PCR analysis. FACS-purified BC CML progenitors (CD34+CD38+Lin-PI−) were transplanted intrahepatically into newborn RAG2−/− c−/− mice and serially transplanted for propagation of tumors. Harvested tumors were then CD34-selected and transplanted to tertiary recipients (1×105 cells per mouse). Mice were treated for 14 days starting at 8 weeks of age. Mice were dosed once daily by oral gavage with 200µl of drug or vehicle (50% PEG in HBSS). The treatment included PF-04449913 (100mg/kg) with or without dasatinib (50mg/kg). After treatment the percentage of LSC (CD34+CD38+CD45RA+CD123+Lin-PI−) was determined by FACS analysis of the bone marrow and liver and number of tumors evaluated. BC CML progenitors harbors an up regulation of Gli mRNA and a downregulation of Ptch1 mRNA (n=3) compared to normal cord blood. Gli mRNA expression decreases following in vitro treatment with PF-04449913. Treatment with PF-04449913 alone in human BC CML LSC transplanted mice (n=3 exp., n=15 mice) does not significantly inhibit engraftment compared with vehicle (n=3 exp., n= 27 mice). Although dasatinib alone decreases tumor formation (P≤0.05), it does not eradicate the LSC in the liver or bone marrow (n=3 exp., n=20 mice). Combination treatment with PF-04449913 and dasatinib revealed a significant decrease in LSC engraftment in livers (P≤0.05) compared with the PF-04449913 or dasatinib alone. The combination treatment completely eradicated the LSC capability of tumor formation (0.0) in secondary recipients (n=5) compared with dasatinib that had an average of 1.8 tumors (n=6), PF-04449913 that had an average of 4.9 (n=7) and vehicle group that had 3.8 tumors (n=13). Colony formation assays were performed with cord blood CD34+ progenitors (n=3 samples) and no toxicity could be seen with PF-04449913 (1µM) compared with vehicle. There was no significant inhibition of normal CD34+ colony replating following PF-04449913 compared with vehicle treatment. A phase I clinical trial with PF-04449913 in combination with dasatinib is currently being executed for advanced phase CML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-258.

Abstract 1627: Requirement for the Src family kinase, Fyn, in proliferation of BCR-ABL1 transduced bone marrow and aneuploidy in CML

Abstract Oncogenic signals stemming from the BCR/ABL kinase drive the development and progression of chronic myeloid leukemia (CML). Hence, therapies blocking kinase activity have proven extremely successful at managing this hematological malignancy. However, treatment of the terminal and progressed state of CML, blast crisis, remains difficult. Previous work from our group has identified the Src family kinase, Fyn, as being upregulated in blast crisis CML. We also reported that knockdown of Fyn slowed CML growth in vitro and in vivo and sensitized cells to BCR-ABL1 kinase inhibition with imatinib. Recent work using microarray profiling of kinase inhibitor resistant cells from a separate group has shown that imatinib resistance is associated with Fyn expression. In the current study, we utilized bone marrow cells from Fyn deficient mice to test the requirement for Fyn in p210 BCR-ABL1 mediated survival. Bone marrow cells from Fyn knockout and Fyn wildtype mice were harvested and transduced with a GFP expressing retroviral construct (MigR1) or MigR1-p210 BCR-ABL1. Cells were sorted for GFP expression and BCR-ABL1 expression was confirmed by Western blotting. Cell growth in culture was monitored over the course of three weeks in Fyn deficient and wildtype cells expressing BCR-ABL1. Fourteen days post sorting, the number of cells lacking Fyn were more than 50% lower than Fyn wildtype cells transduced with BCR-ABL1. Clonogenic assays further indicated that Fyn significantly promotes BCR-ABL1 induced bone marrow cell growth. Colony numbers in Fyn deficient cells expressing BCR-ABL1 were less than half that seen in Fyn wildtype cells. To address the role for Fyn kinase activity in these effects, constitutively active and dominant negative Fyn constructs have been transduced into BCR-ABL1 expressing cells. Constitutively active Fyn caused a high degree of chromosomal aberrations, fusions and aneuploidy suggestive of genomic instability. Taken together, these data highlight a significant role for Fyn in BCR-ABL1 mediated proliferation and secondary chromosomal aberrations, which are both features characteristic of blast crisis CML. Our results indicate that strategies to inhibit Fyn expression and kinase activity will be therapeutically relevant for progressed and/or refractory CML patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1627.

Abstract 3687: Expression of a Src-family kinase in CML cells induces resistance to imatinib in a kinase-dependent manner

Abstract Chronic Myelogenous Leukemia (CML) is characterized by the presence of Philadelphia Chromosome, which results in the expression of the 210 kDa Bcr-Abl tyrosine kinase. Bcr-Abl constitutively activates signaling pathways important for the proliferation, survival and cytokine independence of myeloid progenitors, including the Src family kinases (SFKs). Imatinib, an inhibitor of Bcr-Abl tyrosine kinase activity, is the frontline therapy for CML. Although imatinib is remarkably effective in the chronic phase of the disease, patients with accelerated or blast crisis CML often develop resistance. Several recent studies performed on clinical specimens from imatinib-resistant patients with wild-type Bcr-Abl found that the Src family kinase members Hck and Lyn are overexpressed or highly active, suggesting that Src kinases may play a role in imatinib resistance. While the role of Lyn has been addressed in some detail, the contribution of Hck to this type of imatinib resistance is less well understood. To test whether Hck overexpression in CML cells induces resistance to imatinib in a kinase-dependent manner, we employed a Hck mutant (Hck-T338A) that is uniquely sensitive to the pyrazolo-pyrimidine inhibitor, NaPP1. In vitro, Hck-T338A was 48 times more sensitive to NaPP1 than the wild-type kinase. Importantly, this “gatekeeper” mutation was functionally silent and did not confer a loss or gain of function on Hck in a control fibroblast transformation assay. Expression of wild-type Hck or the T338A mutant in K562 CML cells resulted in resistance to imatinib-induced apoptosis and inhibition of soft-agar colony formation. Treatment with NaPP1 restored sensitivity to imatinib in a concentration-dependent manner only in cells expressing the Hck-T338A mutant. In contrast, cells expressing wild-type Hck were not affected by NaPP1, demonstrating the selectivity of NaPP1 for the Hck-T338A mutant. This result shows that Hck-induced imatinib resistance requires Hck kinase activity. In addition, NaPP1 also reduced Hck-mediated phosphorylation of Bcr-Abl at sites that may affect imatinib binding exclusively in cells expressing Hck-T338A. Together, these data establish a direct cause and effect relationship between Hck overexpression and imatinib resistance in CML cells. Furthermore, our results show that imatinib resistance requires Hck kinase function and may occur via direct Hck-induced trans-phosphorylation of Bcr-Abl. Selective drug targeting of Hck may be of therapeutic benefit in imatinib-resistant CML patients with increased Hck expression or activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3687.