Probability of treatment‐free remission (TFR) in CML patients with additional chromosomal abnormalities (ACA) in the Philadelphia‐positive clone or variant Philadelphia translocations (ACA/Var‐Ph ...group, blue panel), in those with no cytogenetic abnormality other than the classical Philadelphia translocation (c‐Ph group, green panel) and in the subgroups of CML patients with high‐risk ACA (HR‐ACA, yellow panel) and Var‐Ph (red panel).
Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease due to ...deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wild-type AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without pre-existing immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of pre-existing immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy.
Hereditary spastic paraplegia (HSP) is characterized by the specific retrograde degeneration of the longest axons in the central nervous system, the corticospinal tracts. The gene most frequently ...involved in autosomal dominant cases of this disease, SPG4, encodes spastin, an ATPase belonging to the AAA family. AAA proteins are thought to exert their function by the energy-dependent rearrangement of protein complexes. The composite function of these proteins is directed by their binding to regulatory factors and adaptor proteins that target their activity into specific pathways in vivo. We previously found that overexpressed spastin interacts dynamically with microtubules and displays microtubule-severing activity. Here, we demonstrate that spastin is enriched in cell regions containing dynamic microtubules. During cell division spastin is found in the spindle pole, the central spindle and the midbody, whereas in immortalized motoneurons it is enriched in the distal axon and the branching points. Furthermore, spastin interacts with the centrosomal protein NA14, and co-fractionates with γ-tubulin, a centrosomal marker. Deletion of the region required for binding to NA14 disrupts spastin interaction with microtubules, suggesting that NA14 may be an important adaptor to target spastin activity at the centrosome. These data strongly argue that spastin plays a role in cytoskeletal rearrangements and dynamics, and provide an attractive explanation for the degeneration of motor axons in HSP.
Introduction: As patients (pts) diagnosed with chronic myeloid leukaemia (CML) in CP are predicted to have a life expectancy comparable to that of the normal population, clinical concern has focused ...on the burden of long-term side effects and quality of life. Stopping tyrosine kinase inhibitor (TKI) is possible in a selected group of pts, resulting in a 50% chance of treatment free remission (TFR). Limited data, however, are available on the outcome of TKI dose reduction (DR) in maintaining molecular responses.
Methods: We retrospectively analysed the outcome of TKI DR in pts in ≥MR3 treated at our centre from Jan 2000 until May 2015. We defined different low dose groups (LDG), according to the actual TKI dose: for imatinib (IM), 300mg and 200mg; for dasatinib (DAS), 70-80mg, 50mg, 40mg and 20mg; for nilotinib (NIL), 400-450mg, 300mg or ≤200mg; and for bosutinib (BOS), 300mg, 200mg and <200mg.
Given the ‘real life’ setting, pts may have received either 1) multiple DRs of the same TKI or 2) different TKIs at different low doses. For scenario 1): we analysed the dose that maintained ≥MR3 and was used for the longest period of time. In case of loss of molecular response on a lower dose level, the next dose was considered a further ‘case’ and the patient was analysed more than once. In scenario 2) the patient was analysed once for each TKI received at low dose. MR3 and MR4 were defined conventionally. The molecular recurrence free survival (MRFS) was estimated by Kaplan-Meier.
Results: We included 232 pts (IM=83 pts, cases=85; DAS=75, cases=79; NIL=72, cases=73; BOS=32, cases=33), of whom 8 pts were included in two different LDG on the same TKI (because of loss of response on the lower dose: imatinib n=2, dasatinib n=4, nilotinib n=1, bosutinib n=1). 22 and 4 pts received 2 low dose (LD) TKIs and 3 LD TKIs respectively. The total number of cases was 270. Reasons for DR included any degree of adverse event deemed significant by the clinician or pre-emptive DR at the time of introduction of a subsequent TKI due to intolerance to the previous TKI. All IM pts were treated first line, whereas the majority of pts (n=159, 88.8%) received their current 2GTKI as ≥ 2 line. Median follow-up on LD TKI was 25.3 months (1.9-175). Patient characteristics by TKI are shown in Tables 1-4.
The 2-year MRFS were: IM, 88.4% (95% CI, 87.7-89.1%), and 92.7% and 77.2% for LDG1 and LDG2 respectively; DAS 92.8% (95% CI, 92.2-93.4%); and 100%, 96.2%, 92.3% and 85.6% for LDG1, LDG2, LDG3 and LDG4 respectively; NIL 93.4% (95% CI, 92.6-94.1%); and 93.3%, 88.9% and 100% for LDG1, LDG2 and LDG3, respectively; BOS 91.7% (95% CI, 90-93%); and 100% for LDG1 and LDG2 and 75% for LDG3 (Figure 1 a,b,c and d).
One patient on NIL required DR for grade 3 liver toxicity, progressed to blast crisis after losing MR3 on 300mg daily and died post allo-SCT. One patient, who had achieved only CHR on IM, developed a T315I mutation on 50mg DAS second line while in MR3, having lost MR4, and was changed to ponatinib. One patient on DAS died of an unrelated brain tumour.
In each TKI cohort, 59/83 pts (71%) remained on LD IM, 51/75 (68%) pts on LD DAS, 35/72 (48.6%) pts on LD NIL and 30/32 (93.7%) pts on LD BOS. 55 pts stopped LD TKI while in sustained MR4 or greater (IM n=21/83 25.3%, DAS n=12/75 16%, NIL n=20/72 27.8%, BOS n=2/30 6.6%) with a 2-year probability of TFR of 79.4% (95% CI, 78.3-80.5%) (compared to 50% at 2 years in EURO-SKI), with a median observation time of 28 months (5-83.7) in non-relapsing pts. TFR in the different cohorts were 85.7%, 62.3%, 80% and 100% for IM, DAS, NIL and BOS respectively.
Conclusion: For selected pts in ≥MR3 lowering the TKI dose can improve the tolerability of TKI therapy without impacting responses. The higher rate of TFR observed in our pts than in published stopping studies probably reflects cohorts of pts already shown to maintain deep responses on lower than standard doses of TKI, and mirrors the results of the UK NIHR Destiny study.
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Apperley:Incyte: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Milojkovic:Incyte: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau.
As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or ...significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients.
Summary
Few effective therapies exist for acute myeloid leukaemia (AML), in part due to the molecular heterogeneity of this disease. We sought to identify genes crucial to deregulated AML signal ...transduction pathways which, if inhibited, could effectively eradicate leukaemia stem cells. Due to difficulties in screening primary cells, most previous studies have performed next‐generation sequencing (NGS) library knockdown screens in cell lines. Using carefully considered methods including evaluation at multiple timepoints to ensure equitable gene knockdown, we employed a large NGS short hairpin RNA (shRNA) knockdown screen of nearly 5 000 genes in primary AML cells from six patients to identify genes that are crucial for leukaemic survival. Across various levels of stringency, genome‐wide bioinformatic analysis identified a gene in the NOX family, NOX1, to have the most consistent knockdown effectiveness in primary cells (P = 5∙39 × 10−5, Bonferroni‐adjusted), impacting leukaemia cell survival as the top‐ranked gene for two of the six AML patients and also showing high effectiveness in three of the other four patients. Further investigation of this pathway highlighted NOX2 as the member of the NOX family with clear knockdown efficacy. We conclude that genes in the NOX family are enticing candidates for therapeutic development in AML.
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