Global down-regulation of HOX gene expression in PML-RARalpha + acute promyelocytic leukemia identified by small-array real-time PCR

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex network of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha fusion proteins have been reported to act as part of a repressor complex during myeloid cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.

Entinostat prevents leukemia maintenance in a collaborating oncogene-dependent model of CN-AML.

The incidence of refractory acute myeloid leukemia (AML) is on the increase due in part to an aging population that fails to respond to traditional therapies. High throughput genomic analysis promises better diagnosis, prognosis and therapeutic intervention based on improved patient stratification. Relevant pre-clinical models are urgently required to advance drug development in this area. The collaborating oncogenes, HOXA9 and MEIS1, are frequently co-overexpressed in cytogenetically normal AML (CN-AML) and a conditional transplantation mouse model was developed that demonstrated oncogene-dependency and expression levels comparable to CN-AML patients. Integration of gene signatures obtained from the mouse model and a cohort of CN-AML patients using statistically significant connectivity Map (sscMap) analysis identified Entinostat as a drug with the potential to alter the leukemic condition towards the normal state. Ex vivo treatment of leukemic cells, but not age-matched normal bone marrow controls, with Entinostat validated the gene signature and resulted in reduced viability in liquid culture, impaired colony formation and loss of the leukemia initiating cell. Furthermore, in vivo treatment with Entinostat resulted in prolonged survival of leukemic mice. This study demonstrates that the HDAC inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML. © 2013 AlphaMed Press

Distinct poor prognostic subgroups of acute myeloid leukaemia, FLT3-ITD and P-glycoprotein-positive, have contrasting levels of FOXO1

Regulation of ABCB1 (P-glycoprotein/Pgp) in AML was investigated. In a historical cohort with Pgp and transcriptional regulator expression profiling data available (n=141), FOXO1 correlated with Pgp protein expression. This was confirmed in an independent cohort (n=204). Down-regulation (siRNA) or hyperactivation (nicotinamide) of FOXO1 led to corresponding changes in Pgp. Low FOXO1 expression correlated with FLT3-ITDs (p

The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia

Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1–CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3 (rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05). Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1–CCN3 signalling contributes to the pathogenesis of CML.

Risk of myeloid neoplasms after solid organ transplantation

Solid organ transplant recipients have elevated cancer risks, owing in part to pharmacologic immunosuppression. However, little is known about risks for hematologic malignancies of myeloid origin. We linked the US Scientific Registry of Transplant Recipients with 15 population-based cancer registries to ascertain cancer occurrence among 207 859 solid organ transplants (1987–2009). Solid organ transplant recipients had a significantly elevated risk for myeloid neoplasms, with standardized incidence ratios (SIRs) of 4.6 (95% confidence interval 3.8–5.6; N=101) for myelodysplastic syndromes (MDS), 2.7 (2.2–3.2; N=125) for acute myeloid leukemia (AML), 2.3 (1.6–3.2; N=36) for chronic myeloid leukemia and 7.2 (5.4–9.3; N=57) for polycythemia vera. SIRs were highest among younger individuals and varied by time since transplantation and organ type (Poisson regression P<0.05 for all comparisons). Azathioprine for initial maintenance immunosuppression increased risk for MDS (P=0.0002) and AML (2–5 years after transplantation, P=0.0163). Overall survival following AML/MDS among transplant recipients was inferior to that of similar patients reported to US cancer registries (log-rank P<0.0001). Our novel finding of increased risks for specific myeloid neoplasms after solid organ transplantation supports a role for immune dysfunction in myeloid neoplasm etiology. The increased risks and inferior survival should heighten clinician awareness of myeloid neoplasms during follow-up of transplant recipients.

Sequential treatment with flavopiridol synergistically enhances pyrrolo-1,5-benzoxazepine-induced apoptosis in human chronic myeloid leukaemia cells including those resistant to imatinib treatment

The Bcr-Abl kinase inhibitor, imatinib mesylate, is the front line treatment for chronic myeloid leukaemia (CML), but the emergence of imatinib resistance has led to the search for alternative drug treatments and the examination of combination therapies to overcome imatinib resistance. The pro-apoptotic PBOX compounds are a recently developed novel series of microtubule targeting agents (MTAs) that depolymerise tubulin. Recent data demonstrating enhanced MTA-induced tumour cell apoptosis upon combination with the cyclin dependent kinase (CDK)-1 inhibitor flavopiridol prompted us to examine whether this compound could similarly enhance the effect of the PBOX compounds. We thus characterised the apoptotic and cell cycle events associated with combination therapy of the PBOX compounds and flavopiridol and results showed a sequence dependent, synergistic enhancement of apoptosis in CML cells including those expressing the imatinib-resistant T315I mutant. Flavopiridol reduced the number of polyploid cells formed in response to PBOX treatment but only to a small extent, suggesting that inhibition of endoreplication was unlikely to play a major role in the mechanism by which flavopiridol synergistically enhanced PBOX-induced apoptosis. The addition of flavopiridol following PBOX-6 treatment did however result in an accelerated exit from the G2/M transition accompanied by an enhanced downregulation and deactivation of the CDK1/cyclin B1 complex and an enhanced degradation of the inhibitor of apoptosis protein (IAP) survivin. In conclusion, results from this study highlight the potential of these novel series of PBOX compounds, alone or in sequential combination with flavopiridol, as an effective therapy against CML.

The novel pyrrolo-1,5-benzoxazepine, PBOX-21, potentiates the apoptotic efficacy of STI571 (imatinib mesylate) in human chronic myeloid leukaemia cells

The Bcr-Abl kinase inhibitor, STI571, is the first line treatment for chronic myeloid leukaemia (CML), but the recent emergence of STI571 resistance has led to the examination of combination therapies. In this report, we describe how a novel non-toxic G1-arresting compound, pyrrolo-1,5-benzoxazepine (PBOX)-21, potentiates the apoptotic ability of STI571 in Bcr-Abl-positive CML cells. Co-treatment of CML cells with PBOX-21 and STI571 induced more apoptosis than either drug alone in parental (K562S and LAMA84) and STI571-resistant cells lines (K562R). This potentiation of apoptosis was specific to Bcr-Abl-positive leukaemia cells with no effect observed on Bcr-Abl-negative HL-60 acute myeloid leukaemia cells. Apoptosis induced by PBOX-21/STI571 resulted in activation of caspase-8, cleavage of PARP and Bcl-2, upregulation of the pro-apoptotic protein Bim and a downregulation of Bcr-Abl. Repression of proteins involved in Bcr-Abl transformation, the anti-apoptotic proteins Mcl-1 and Bcl-(XL) was also observed. The combined lack of an early change in mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9 suggests that this pathway is not involved in the initiation of apoptosis by PBOX-21/STI571. Apoptosis was significantly reduced following pre-treatment with either the general caspase inhibitor Boc-FMK or the chymotrypsin-like serine protease inhibitor TPCK, but was completely abrogated following pre-treatment with a combination of these inhibitors. This demonstrates the important role for each of these protease families in this apoptotic pathway. In conclusion, our data highlights the potential of PBOX-21 in combination with STI571 as an effective therapy against CML.

Marrow aplasia developing 13 years after HLA-identical sibling allogeneic transplantation for chronic myeloid leukaemia:successful treatment with antithymocyte globulin and peripheral blood stem cell infusion from the original donor

Secondary or late graft failure has been defined as the development of inadequate marrow function after initial engraftment has been achieved. We describe a case of profound marrow aplasia occurring 13 years after sibling allogeneic bone marrow transplantation for chronic myeloid leukaemia (CML) in first chronic phase. Although the patient remained a complete donor chimera, thereby suggesting that an unselected infusion of donor peripheral blood stem cells (PBSC) or bone marrow might be indicated, the newly acquired aplasia was thought to be immune in aetiology and some immunosuppression was therefore considered appropriate. Rapid haematological recovery was achieved after the infusion of unselected PBSC from the original donor following conditioning with anti-thymocyte globulin (ATG).

Immune haemolytic anaemia following T cell-depleted allogeneic bone marrow transplantation for chronic myeloid leukaemia:association with leukaemic relapse and treatment with donor lymphocyte infusions

Immune haemolytic anaemia (IHA) is a recognised complication after allogeneic stem cell transplantation (SCT) and occurs more frequently if marrow cells have been subjected to T cell depletion (TCD). Among 58 consecutive patients who underwent TCD-allogeneic SCT from volunteer unrelated donors for the treatment of CML at the Hammersmith Hospital during a 3-year period (1 March 1996 to 28 February 1999) we identified nine cases of IHA. All patients had a strongly positive direct and indirect antiglobulin test and in eight patients the serological findings were typical of warm-type haemolysis often with antibody specificities within the Rh system. All nine cases had clinically significant haemolysis and were treated initially with prednisolone and immunoglobulin. The onset of IHA coincided with the occurrence of leukaemic relapse in six cases, and the presence of host haemopoiesis confirmed by lineage-specific chimerism in all four cases studied. Five patients received donor lymphocyte infusions (DLI); in three molecular remission and the restoration of full donor chimerism coincided with resolution of haemolysis. We conclude that in the context of leukaemic relapse, DLI is an effective therapy for IHA following allografts involving TCD.

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia

Donor lymphocyte infusions (DLI) have been shown to enhance the graft-versus-leukaemia (GVL) effect and induce haematological and molecular remission in patients with relapsed CML following allogeneic bone marrow transplantation (BMT). The potent donor cell-mediated cytolysis following DLI may lead to a short period of aplasia before the re-establishment of donor haematopoiesis. The absence of detectable donor cells in patients prior to DLI infusion may result in permanent aplasia in certain patients. We report on four patients who relapsed 1, 3, 6.5 and 7 years post-BMT for chronic phase CML and were treated with DLI from their original BMT donor. Polymorphic short tandem repeats (STRs) were used to assess haematological chimaerism both prior to and following DLI. At the time of relapse, STR-PCR indicated the presence of donor cells in all four patients, at levels ranging from 1-40%. A clinical and molecular response was seen in 4/4 patients following a short period of cytopenia and all patients remain in clinical remission with a follow-up of 2 months-3 years post-DLI. STR-PCR indicated that a response was occurring during the period of pancytopenia when metaphase analysis was unsuccessful. Lineage-specific analysis of the cellular response to DLI was monitored using STR-PCR of peripheral blood (PB) and bone marrow (BM) lymphocyte-enriched fractions and CD2-positive and -negative T cell fractions. In one patient BM and PB CD34-positive and -negative fractions were also assessed. A change in the ratio of donor:recipient cells in the PB lymphocyte fraction was the earliest molecular indication of an anti-leukaemic response. Subsequent conversion to donor chimaerism occurred in the other lineages and the granulocyte fraction was the last lineage to convert. In conclusion, lineage-specific STR-PCR permits detailed monitoring of subtle changes in donor/recipient cell dynamics in specific lineages following DLI during the crucial pancytopenic phase and may be a useful predictor of haematological response to DLI therapy.

Persistent donor chimaerism is consistent with disease-free survival following BMT for chronic myeloid leukaemia

Chronic myeloid leukaemia (CML) can be treated successfully with allogeneic bone marrow transplantation (BMT) leading to long-term disease-free survival. Leukemia relapse, however, remains a significant clinical problem. Relapse following BMT presumably results from the expansion of small numbers of recipient leukaemic cells which have survived the conditioning therapy. In order to define patients who are at a high risk of leukaemia relapse, a variety of techniques have been employed to detect persistence of host haemopoiesis (mixed chimaerism, MC) or residual leukaemia (minimal residual disease, MRD). However, the precise relationship between the detection of MC and MRD post-BMT is unknown. We have investigated chimaerism and MRD status in 22 patients who were in clinical and haematological remission post-allogeneic BMT for chronic phase CML. Chimaerism was assessed using short tandem repeat PCR (STR-PCR) while BCR-ABL mRNA detection using reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the presence of MRD. Seventeen patients received unmanipulated marrow (non-TCD) while in five patients a T cell-depleted transplant (TCD) was performed as additional GVHD prophylaxis. Chimaerism was evaluated in 18 patients (14 non-TCD, four TCD). Mixed chimaerism was an uncommon finding in recipients of unmanipulated BMT (21%) when compared to TCD BMT (100%). No evidence of MRD, as identified using the BCR-ABL mRNA RT-PCR assay, was detected in those patients who were donor chimaeras. Early and transient MC and MRD was detected in four patients (two non-TCD, two TCD) who have subsequently converted to a donor profile. One patient has stable low-level MC but remains MRD negative 4 years post-BMT. Late MC and MRD was observed in two patients who relapsed >6 years after TCD BMT for CML. We conclude that mixed chimaerism is a rare event in recipients of unmanipulated BMT and that donor chimaerism as detected by STR-PCR assay is consistent with disease-free survival and identifies patients with a low risk of leukaemic relapse post-BMT for CML.

Donor chimaerism is a strong indicator of disease free survival following bone marrow transplantation for chronic myeloid leukaemia

Although Chronic Myeloid Leukaemia (CML) can be treated successfully with allogeneic bone marrow transplantation (BMT), leukaemia relapse remains a significant clinical problem. Molecular monitoring of the post transplant marrow can be useful in predicting relapse particularly in CML patients where the Philadelphia chromosome or its molecular counterpart, the BCR-ABL fusion messenger RNA can be used as a leukaemia specific marker of minimal residual disease (MRD). We have investigated chimaerism (using polymerase chain reaction of short tandem repeat sequences (STR-PCR)) and MRD status (using reverse transcriptase PCR of the BCR-ABL fusion mRNA) in a serial fashion in 18 patients who were in clinical and haematological remission post allogeneic BMT for chronic phase CML. Eleven patients exhibited complete donor chimaerism with no evidence of minimal residual disease. Five patients had transient or low level stable MC. Late MC and MRD was observed in two patients who relapsed > 6 years after T cell depleted BMT for CML. Thus STR-PCR is an appropriate screening test in the post transplant setting for CML patients, but those patients exhibiting mixed haemopoietic chimaerism should also be monitored using a leukaemia specific sensitive molecular assay.

Successful second unrelated donor BMT in a child with juvenile chronic myeloid leukaemia:documentation of chimaerism using the polymerase chain reaction

A 3-year old child with juvenile chronic myeloid leukaemia received a T cell-depleted BMT from a male unrelated donor. There was early graft failure associated with increasing splenomegaly and hypersplenism. Splenectomy was performed 53 days post-transplant and was followed by autologous marrow recovery with return of leukaemia. A second unrelated donor BMT was performed 9 months later using T cell-replete marrow from a similarly matched female donor. Grade 2 GVHD involving the skin and gut responded to treatment with steroids. Chimaerism was assessed using Y-specific polymerase chain reaction (PCR) and microsatellites. Samples taken at the time of splenectomy showed no donor marrow engraftment but there was significant engraftment in the spleen. Following the second transplant, donor-type haematopoiesis was documented using a panel of microsatellite probes. The patient remains well 6 months after transplant. Splenectomy should be considered prior to transplant in patients with significant splenomegaly and hypersplenism. Partial chimaerism in the spleen, but not bone marrow, post-BMT, has not previously been documented. PCR technology is a useful and highly sensitive way to assess chimaerism post-BMT and is informative in sex-matched cases, whilst the small amount of material required is advantageous in paediatric patients.

CD33 reponses are blocked by SOCS3 through accelerated proteasomal-mediated turnover

CD33 is a member of the sialic acid–binding immunoglobulin-like lectin (Siglec) family of inhibitory receptors and a therapeutic target for acute myeloid leukemia (AML). CD33 contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), which can recruit SHP-1 and SHP-2. How CD33 expression is regulated is unclear. Suppressor of cytokine signaling 3 (SOCS3) is expressed in response to cytokines, LPS, and other PAMPs, and competes with SHP-1/2 binding to ITIMs of cytokine receptors, thereby inhibiting signaling. In this study, using peptide pull-down experiments, we found that SOCS3 can specifically bind to the phosphorylated ITIM of CD33. Additionally, following cross-linking SOCS3 can recruit the ECS E3 ligase resulting in accelerated proteasomal degradation of both CD33 and SOCS3. Our data suggest that the tyrosine motifs in CD33 are not important for internalization, while they are required for degradation. Moreover, SOCS3 inhibited the CD33-induced block on cytokine-induced proliferation. This is the first receptor shown to be degraded by SOCS3 and where SOCS3 and its target protein are degraded concomitantly. Our findings clearly suggest that during an inflammatory response, the inhibitory receptor CD33 is lost by this mechanism. Moreover, this has important clinical implications as tumors expressing SOCS3 may be refractory to -CD33 therapy.