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The treatment of blast phase chronic myeloid leukemia (bpCML) remains a challenge due at least in part to drug resistance of leukemia stem cells (LSCs). Recent clinical evidence suggests that the BCL-2 inhibitor venetoclax in combination with ABL-targeting tyrosine kinase inhibitors (TKIs) can eradicate bpCML LSCs. In this report, we employed preclinical models of bpCML to investigate the efficacy and underlying mechanism of LSC-targeting with venetoclax/TKI combinations. Transcriptional analysis of LSCs exposed to venetoclax and dasatinib revealed upregulation of genes involved in lysosomal biology, in particular lysosomal acid lipase A (LIPA), a regulator of free fatty acids. Metabolomic analysis confirmed increased levels of free fatty acids in response to venetoclax/dasatinib. Pre-treatment of leukemia cells with bafilomycin, a specific lysosome inhibitor, or genetic perturbation of LIPA, resulted in increased sensitivity of leukemia cells toward venetoclax/dasatinib, implicating LIPA in treatment resistance. Importantly, venetoclax/dasatinib treatment does not affect normal stem cell function, suggestive of a leukemia-specific response. These results demonstrate that venetoclax/dasatinib is an LSCselective regimen in bpCML and that disrupting LIPA and fatty acid transport enhances venetoclax/dasatinib response in targeting LSCs, providing a rationale for exploring lysosomal disruption as an adjunct therapeutic strategy to prolong disease remission.

Venetoclax+azacitidine is the standard of care for newly-diagnosed patients with acute myeloid leukemia (AML) for whom intensive chemotherapy is inappropriate. Efforts to optimize this regimen are necessary. We designed a clinical trial to investigate two hypotheses: i) higher doses of venetoclax are tolerable and more effective, and ii) azacitidine can be discontinued after deep remissions. Forty-two newly diagnosed AML patients were enrolled in the investigator-initiated High Dose Discontinuation Azacitidine+Venetoclax (HiDDAV) Study (clinicaltrials gov. Identifier: NCT03466294). Patients received one to three “induction” cycles of venetoclax 600 mg daily with azacitidine. Responders received MRD-positive or MRDnegative “maintenance” arms: azacitidine with 400 mg venetoclax or 400 mg venetoclax alone, respectively. The toxicity profile of HiDDAV was similar to 400 mg venetoclax. The overall response rate was 66.7%; the duration of response (DOR), event-free survival (EFS) and overall survival were 12.9, 7.8 and 9.8 months, respectively. The MRD negativity rate was 64.3% by flow cytometry and 25.0% when also measured by droplet digital polymerase chain recation. MRD-negative patients by flow cytometry had improved DOR and EFS; more stringent measures of MRD negativity were not associated with improved OS, DOR or EFS. Using MRD to guide azacitidine discontinuation did not lead to improved DOR, EFS or OS compared to patients who discontinued azacitidine without MRD guidance. Within the context of this study design, venetoclax doses >400 mg with azacitidine were well tolerated but not associated with discernible clinical improvement, and MRD may not assist in recommendations to discontinue azacitidine. Other strategies to optimize, and for some patients, de-intensify, venetoclax+azacitidine regimens are needed.

Lévy flight is a type of random walk that characterizes the behaviour of many natural phenomena studied across a multiplicity of academic disciplines; within biology specifically, the behaviour of fish, birds, insects, mollusks, bacteria, plants, slime molds, t-cells, and human populations. The Lévy flight foraging hypothesis states that because Lévy flights can maximize an organism's search efficiency, natural selection should result in Lévy-like behaviour. Empirical and theoretical research has provided ample evidence of Lévy walks in both extinct and extant species, and its efficiency across models with a diversity of resource distributions. However, no model has addressed the maintenance of Lévy flight foraging through evolutionary processes, and existing models lack ecological breadth. We use numerical simulations, including lineage-based models of evolution with a distribution of move lengths as a variable and heritable trait, to test the Lévy flight foraging hypothesis. We include biological and ecological contexts such as population size, searching costs, lifespan, resource distribution, speed, and consider both energy accumulated at the end of a lifespan and averaged over a lifespan. We demonstrate that selection often results in Lévy-like behaviour, although conditional; smaller populations, longer searches, and low searching costs increase the fitness of Lévy-like behaviour relative to Brownian behaviour. Interestingly, our results also evidence a bet-hedging strategy; Lévy-like behaviour reduces fitness variance, thus maximizing geometric mean fitness over multiple generations.

Phantom limb pain is commonly known as a neurological condition, where an amputee will continue to feel a limb that is no longer present in a painful fashion. Virtual mirror therapy (VMT) has been suggested as a method for alleviating phantom limb pain. The inclusion of tactile sensation in VMT has shown to be beneficial; however, delivering a tactile sensation to a phantom limb, without the use of invasive procedures, can be difficult. The current approach for transferring a tactile sensation to a phantom limb is called visual capture. The ability to establish visual capture has been demonstrated in VMT applications. However, there is little research into whether an established visual capture effect can be relocated to a more distal location for phantom limb pain management. This paper investigates whether a passive vibrotactile sensation can be moved to a distal location from its veridical location using a series of distally located lights presented in either a random or a structured fashion. Eight non-amputee participants were tasked with localising a static tactile sensation on a virtual arm. These vibrotactile sensations were presented simultaneously with a visual light stimulus, either co-located or located distally at three different locations. Findings show that a tactile sensation without a visual stimulus was difficult for participants to localise; however, when a visual stimulus was added, they were better able to locate the veridical tactile position. The structured group exhibited a larger range of tactile relocation responses than the random group. However, this result was unreliable, with the majority of the responses situated at the vibrotactile actuator. There was a significant difference between the random and structured group’s ability to retain a visual capture at the veridical vibrotactile location when the lights were located distally. The random group did not express a visual capture response when the lights were presented distally while the structured group did, suggesting the structured group developed a more robust association between the visual stimulus and the vibrotactile stimulus. Findings may be of use where increasing tactile acuity without significant alteration of a veridical location is a desired therapeutic outcome.

Virtual reality is increasingly used in rehabilitation and can provide additional motivation when working toward therapeutic goals. However, a particular problem for patients regards their ability to plan routes in unfamiliar environments. Therefore, the aim of this study was to explore how visual cues, namely embedded context-sensitive attractors, can guide attention and walking direction in VR, for clinical walking interventions. This study was designed using a butterfly as the embedded context-sensitive attractor, to guide participant locomotion around the clinical figure of eight walk test, to limit the use of verbal instructions. We investigated the effect of varying the number of attractors for figure of eight path following, and whether there are any negative impacts on perceived autonomy or workload. A total of 24 participants took part in the study and completed six attractor conditions in a counterbalanced order. They also experienced a control VE (no attractors) at the beginning and end of the protocol. Each VE condition lasted a duration of 1 min and manipulated the number of attractors to either singular or multiple alongside, the placement of turning markers (virtual trees) used to represent the cones used in clinical settings for the figure of eight walk test. Results suggested that embedded context-sensitive attractors can be used to guide walking direction, following a figure of eight in VR without impacting perceived autonomy, and workload. However, there appears to be a saturation point, with regards to effectiveness of attractors. Too few objects in a VE may reduce feelings of intrinsic motivation, and too many objects in a VE may reduce the effectiveness of attractors for guiding individuals along a figure of eight path. We conclude by indicating future research directions, for attractors and their use as a guide for walking direction.

ETV6 is an ETS family transcription factor that plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that led to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation was also reflected in patient-derived platelet transcripts and drove aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction.

OBJECTIVES/GOALS: The objective of this study is to define the molecular mechanisms that control survival of malignant stem cells in acute myeloid leukemia (AML). Leukemia stem cells (LSCs) are not effectively eradicated by standard treatment and lead to resistance and relapse, which contribute to poor survival rates. METHODS/STUDY POPULATION: The recently FDA approved venetoclax, a BCL2 inhibitor, with azacitidine, a hypomethylating agent leads to a 70% response rate in AML patients. Analysis of patients treated with this regimen showed direct targeting of LSCs. BCL2 has a non-canonical function in regulation of intracellular calcium. To determine how BCL2 mediated calcium signaling plays a role in LSC biology, we used LSCs isolated from venetoclax/azacitidine (ven/aza) sensitive and resistant patient samples to measure expression of calcium channels via RNA seq. BIO-ID, siRNA, flow cytometry, seahorse assays, calcium measurements and colony assays were used to determine the effects of calcium channel perturbation on LSC biology. RESULTS/ANTICIPATED RESULTS: BCL2 inhibition leads to decreased OXPHOS activity in primary AML specimens. BIO-ID studies revealed cation/metal ion transporters, ER membrane proteins and ER membrane organization as top enriched pathways interacting with BCL2. RNA-seq data showed increased expression of genes involved in calcium influx into the ER in ven/aza sensitive LSCs and increased expression of genes involved in calcium efflux from the ER in ven/aza resistant samples. Ven/Aza resistant LSCs have increased mitochondrial calcium content, consistent with their increased OXPHOS activity as calcium is required for OXPHOS. Perturbation of these channels leads to decreased OXPHOS activity and decreased viability in LSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: We postulate that a deeper understanding of the mechanisms behind ven/aza targeting of LSCs will lead to the development of novel therapies for patients who do not respond to ven/aza. Our data show targeting intracellular calcium signaling could be a viable therapeutic strategy for AML patients.

A graph is locally irregular if every two adjacent vertices have distinct degrees. Recently, Baudon et al. introduced the notion of decomposition into locally irregular subgraphs. They conjectured that for almost every graph $G$, there exists a minimum integer $\chi^{\prime}_{\mathrm{irr}}(G)$ such that $G$ admits an edge-partition into $\chi^{\prime}_{\mathrm{irr}}(G)$ classes, each of which induces a locally irregular graph. In particular, they conjectured that $\chi^{\prime}_{\mathrm{irr}}(G) \leq 3$ for every $G$, unless $G$ belongs to a well-characterized family of non-decomposable graphs. This conjecture is far from being settled, as notably (1) no constant upper bound on$\chi^{\prime}_{\mathrm{irr}}(G)$ is known for $G$ bipartite, and (2) no satisfactory general upper bound on $\chi^{\prime}_{\mathrm{irr}}(G)$ is known. We herein investigate the consequences on this question of allowing a decomposition to include regular components as well. As a main result, we prove that every bipartite graph admits such a decomposition into at most $6$ subgraphs. This result implies that every graph $G$ admits a decomposition into at most $6(\lfloor \mathrm{log} \chi (G) \rfloor +1)$ subgraphs whose components are regular or locally irregular.

Graph Theory

Graphs and Algorithms