Targeting self-renewal pathways in myeloid malignancies

A fundamental property of hematopoietic stem cells (HSCs) is the ability to self-renew. This is a complex process involving multiple signal transduction cascades which control the fine balance between self-renewal and differentiation through transcriptional networks. Key activators/regulators of self-renewal include chemokines, cytokines and morphogens which are expressed in the bone marrow niche, either in a paracrine or autocrine fashion, and modulate stem cell behaviour. Increasing evidence suggests that the downstream signaling pathways induced by these ligands converge at multiple levels providing a degree of redundancy in steady state hematopoiesis. Here we will focus on how these pathways cross-talk to regulate HSC self-renewal highlighting potential therapeutic windows which could be targeted to prevent leukemic stem cell self-renewal in myeloid malignancies

Is there a role for dose modification of TKI therapy in CML

Purpose of review: For patients with chronic phase chronic myeloid leukemia (CP-CML), there is an increasing focus on personalization of therapy with dose modifications of tyrosine kinase inhibitors (TKIs) to reduce side effects and maintain efficacy. Dose reductions are also being considered in clinical trials prior to treatment-free remission (TFR) attempts. Recent findings: Recent retrospective analyses of large clinical trials show that dose modification/reduction is safe. Efficacy is generally maintained and side effects are improved. Clinical trials such as DESTINY have demonstrated that dose reduction is safe for patients in deep molecular remission and may be considered prior to a TFR attempt. Summary: Dose modifications are widely used to prevent and manage the toxicities of TKIs. With adequate monitoring, dose optimization is safe, reduces side effects, and improves quality-of-life for patients. Clinical trials of dose optimization are currently recruiting across all approved TKIs and will lead to further personalization of therapy for CP-CML patients in the future

The generation of transverse and longitudinal vortices in low speed wind tunnels

The present study documents an experimental and numerical investigation into the feasibility of generating longitudinal and transverse vortices in low speed wind tunnels. The longitudinal vortex system is that of a co-rotating vortex pair which, if substituted for a classical single tip vortex, may produce a beneficial modification to Blade Vortex Interaction. The transverse vortex mimics the tip vortex of a typical helicopter rotor and may be used to assess its effect when interacting with other aerofoils or fuselage components. Experiments have been conducted to investigate the flow field associated with two co-rotating vortices which represent the idealised vortex system associated with a novel rotor blade tip platform - the Westland Helicopters Vane Tip. These vortices were generated by two rectangular NACA 0015 half wings positioned upstream of the working section of a low speed wind tunnel. Hot-wire measurements were conducted downstream of the generators using x-wire probes to document the strength, position and size of the vortices. A numerical model was utilised to provide an accurate means of determining vortex strength, position and size. Finally, the model was successfully extended to consider the rotation of the vortex system. The transverse vortex was generated by a rotating blade placed in the contraction of a low speed wind tunnel. A numerical model was utilised in the conceptual design of the experimental facility to model the flow through the settling chamber, contraction, working section and diffuser. This numerical model consisted of a three dimensional source panel method, used to calculate the constrained flow through the low speed tunnel, and a free wake model representing the wake generated by the vortex generator. Convection of the wake was determined by superposition of the undisturbed tunnel velocity and the induced velocity components from the wake itself. Results, obtained via a parametric analysis, illustrate the relationship between the geometry of the wake and basic physical design parameters. On this basis, two possible operational strategies for the upstream rotor are examined with reference to development of the experimental facility. It is concluded that, while a short duration finite rotor traverse may be the optimum vortex generation strategy, a continuous running rotor is a more cost effective and viable option

Bacillus Calmette-Guérin Induces PD-L1 Expression on Antigen-Presenting Cells via Autocrine and Paracrine Interleukin-STAT3 Circuits

Bacillus Calmette-Guérin (BCG) is the only licensed vaccine for tuberculosis (TB), and is also used as an immunotherapy for bladder cancer and other malignancies due to its immunostimulatory properties. Mycobacteria spp., however, are well known for their numerous immune evasion mechanisms that limit the true potential of their therapeutic use. One such major mechanism is the induction of programmed death ligand-1 (PD-L1), which mitigates adaptive immune responses. Here, we sought to unravel the molecular pathways behind PD-L1 up-regulation on antigen-presenting cells (APCs) by BCG. We found that infection of APCs with BCG induced PD-L1 up-regulation, but that this did not depend on direct infection, suggesting a soluble mediator for this effect. BCG induced potent quantities of IL-6 and IL-10, and the downstream transcription factor STAT3 was hyper-phosphorylated. Intracellular analyses revealed that levels of PD-L1 molecules were associated with the STAT3 phosphorylation state, suggesting a causal link. Neutralisation of the IL-6 or IL-10 cytokine receptors dampened STAT3 phosphorylation and BCG-mediated up-regulation of PD-L1 on APCs. Pharmacological inhibition of STAT3 achieved the same effect, confirming an autocrine-paracrine cytokine loop as a mechanism for BCG-mediated up-regulation of PD-L1. Finally, an in vivo immunisation model showed that BCG vaccination under PD-L1 blockade could enhance antigen-specific memory CD4 T-cell responses. These novel findings could lead to refinement of BCG as both a vaccine for infectious disease and as a cancer immunotherapy

Omacetaxine may have a role in chronic myeloid leukaemia eradication through downregulation of Mcl-1 and induction of apoptosis in stem/progenitor cells

Chronic myeloid leukaemia (CML) is maintained by a rare population of tyrosine kinase inhibitor (TKI)-insensitive malignant stem cells. Our long-term aim is to find a BcrAbl-independent drug that can be combined with a TKI to improve overall disease response in chronic-phase CML. Omacetaxine mepesuccinate, a first in class cetaxine, has been evaluated by clinical trials in TKI-insensitive/resistant CML. Omacetaxine inhibits synthesis of anti-apoptotic proteins of the Bcl-2 family, including (myeloid cell leukaemia) Mcl-1, leading to cell death. Omacetaxine effectively induced apoptosis in primary CML stem cells (CD34<sup>+</sup>38<sup>lo</sup>) by downregulation of Mcl-1 protein. In contrast to our previous findings with TKIs, omacetaxine did not accumulate undivided cells <i>in vitro</i>. Furthermore, the functionality of surviving stem cells following omacetaxine exposure was significantly reduced in a dose-dependant manner, as determined by colony forming cell and the more stringent long-term culture initiating cell colony assays. This stem cell-directed activity was not limited to CML stem cells as both normal and non-CML CD34<sup>+</sup> cells were sensitive to inhibition. Thus, although omacetaxine is not leukaemia stem cell specific, its ability to induce apoptosis of leukaemic stem cells distinguishes it from TKIs and creates the potential for a curative strategy for persistent disease

BCR-ABL activity and its response to drugs can be determined in CD34+ CML stem cells by CrkL phosphorylation status using flow cytometry.

In chronic myeloid leukaemia, CD34(+) stem/progenitor cells appear resistant to imatinib mesylate (IM) in vitro and in vivo. To investigate the underlying mechanism(s) of IM resistance, it is essential to quantify Bcr-Abl kinase status at the stem cell level. We developed a flow cytometry method to measure CrkL phosphorylation (P-CrkL) in samples with <10(4) cells. The method was first validated in wild-type (K562) and mutant (BAF3) BCR-ABL(+) as well as BCR-ABL(-) (HL60) cell lines. In response to increasing IM concentration, there was a linear reduction in P-CrkL, which was Bcr-Abl specific and correlated with known resistance. The results were comparable to those from Western blotting. The method also proved to be reproducible with small samples of normal and Ph(+) CD34(+) cells and was able to discriminate between Ph(-), sensitive and resistant Ph(+) cells. This assay should now enable investigators to unravel the mechanism(s) of IM resistance in stem cells

Learning from remote decentralised clinical trial experiences:a qualitative analysis of interviews with trial personnel, patient representatives and other stakeholders

AIMS: The aim of the study was to identify actionable learning points from stakeholders in remote decentralised clinical trials (RDCTs) to inform their future design and conduct. METHODS: Semistructured interviews were carried out with a purposive sample of stakeholders, including senior managers, trial managers, technology experts, principal investigators, clinical investigators, research scientists, research nurses, vendors, patient representatives and project assistants. The interview data were coded using a thematic approach, identifying similarities, differences and clustering to generate descriptive themes. Further refinement of themes was guided by empirical phenomenology, grounding explanation in the meanings that interviewees gave to their experiences. RESULTS: Forty‐eight stakeholders were interviewed. Actionable learning points were generated from the thematic analysis. Patient involvement and participant engagement were seen as critical to the success of RDCTs where in‐person contact is minimal or nonexistent. Involving patients in identifying the research question, creating recruitment materials, apps and websites, and providing ongoing feedback to trial participants were regarded as facilitating recruitment and engagement. Building strong relationships early with trial partners was thought to support RDCT conduct. Multiple modes of capturing information, including patient‐reported outcomes (PROs) and routinely collected data, were felt to contribute to data completeness. However, RDCTs may transfer trial activity burden onto participants and remote‐working research staff, therefore additional support may be needed. CONCLUSION: RDCTs will continue to face challenges in implementing novel technologies. However, maximising patient and partner involvement, reducing participant and staff burden, and simplifying how participants and staff interact with the RDCT may facilitate their implementation

Holographic Thermalization

Using the AdS/CFT correspondence, we probe the scale-dependence of thermalization in strongly coupled field theories following a quench, via calculations of two-point functions, Wilson loops and entanglement entropy in d=2,3,4. In the saddlepoint approximation these probes are computed in AdS space in terms of invariant geometric objects - geodesics, minimal surfaces and minimal volumes. Our calculations for two-dimensional field theories are analytical. In our strongly coupled setting, all probes in all dimensions share certain universal features in their thermalization: (1) a slight delay in the onset of thermalization, (2) an apparent non-analyticity at the endpoint of thermalization, (3) top-down thermalization where the UV thermalizes first. For homogeneous initial conditions the entanglement entropy thermalizes slowest, and sets a timescale for equilibration that saturates a causality bound over the range of scales studied. The growth rate of entanglement entropy density is nearly volume-independent for small volumes, but slows for larger volumes.Comment: 39 pages, 24 figure

MTSS1 is a critical epigenetically regulated tumor suppressor in CML

Chronic myeloid leukemia (CML) is driven by malignant stem cells that can persist despite therapy. We have identified Metastasis suppressor 1 (Mtss1/MIM) to be downregulated in hematopoietic stem and progenitor cells from leukemic transgenic SCLtTA/Bcr-Abl mice and in patients with CML at diagnosis, and Mtss1 was restored when patients achieved complete remission. Forced expression of Mtss1 decreased clonogenic capacity and motility of murine myeloid progenitor cells and reduced tumor growth. Viral transduction of Mtss1 into lineage depleted SCLtTA/Bcr-Abl bone marrow cells decreased leukemic cell burden in recipients, and leukemogenesis was reduced upon injection of Mtss1 overexpressing murine myeloid 32D cells. Tyrosine kinase inhibitor (TKI) therapy and reversion of Bcr-Abl expression increased Mtss1 expression but failed to restore it to control levels. CML patient samples revealed higher DNA methylation of specific Mtss1 promoter CpG sites that contain binding sites for Kaiso and Rest transcription factors. In summary, we identified a novel tumor suppressor in CML stem cells that is downregulated by both Bcr-Abl kinase-dependent and -independent mechanisms. Restored Mtss1 expression markedly inhibits primitive leukemic cell biology in vivo, providing a therapeutic rationale for the Bcr-Abl-Mtss1 axis to target TKI resistant CML stem cells in patients