Lysosomal acid lipase A modulates leukemia stem cell response to venetoclax/tyrosine kinase inhibitor combination therapy in blast phase chronic myeloid leukemia.

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.

MYC Inhibition Potentiates CD8+ T Cells Against Multiple Myeloma and Overcomes Immunomodulatory Drug Resistance.

PURPOSE: Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, are cornerstone therapies in Multiple Myeloma (MM), yet patients inevitably become refractory. IMiDs exert cytotoxicity through inducing Cereblon-dependent proteasomal degradation of IKZF1 and IKZF3, resulting in downregulation of the oncogenic transcription factors IRF4 and MYC. To date, clinical IMiD resistance independent of CRBN or IKZF1/3 has not been well-explored. Here, we investigated the roles of IRF4 and MYC in this context. EXPERIMENTAL DESIGN: Using bone marrow aspirates from patients with IMiD naïve or refractory MM, we examined IKZF1/3 protein levels and IRF4/MYC gene expression following ex vivo pomalidomide treatment via flow cytometry and qPCR. We also assessed ex vivo sensitivity to the MYC inhibitor, MYCi975, using flow cytometry. RESULTS: We discovered that while pomalidomide frequently led to IKZF1/3 degradation in MM cells, MYC gene expression was unaffected by pomalidomide in most IMiD refractory samples. We subsequently demonstrated that MYCi975 exerted strong anti-MM effects in both IMiD naïve and refractory samples. Unexpectedly, we identified CD8+ T cells from patients with MM as crucial effectors of MYCi975-induced cytotoxicity in primary MM samples, and we discovered MYCi975 enhanced the cytotoxic functions of memory CD8+ T cells. We lastly observed synergy between MYCi975 and pomalidomide in IMiD refractory samples, suggesting restoring MYC downregulation can re-sensitize refractory MM to IMiDs. CONCLUSION: Our study supports the concept that MYC represents an Achille's heel in MM across disease states and that MYCi975 may be a promising therapeutic for patients with MM, particularly in combination with IMiDs.

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

Acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL-2, creating a therapeutic opportunity to target LSCs using the BCL-2 inhibitor venetoclax. While venetoclax-based regimens have shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug-responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e. OXPHOS) status with relatively high levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in LSCs and provide an avenue for clinical management of venetoclax resistance.

Intermittent Search, Not Strict Lévy Flight, Evolves under Relaxed Foraging Distribution Constraints.

AbstractThe survival of an animal depends on its success as a forager, and understanding the adaptations that result in successful foraging strategies is an enduring endeavour of behavioral ecology. Random walks are one of the primary mathematical descriptions of foraging behavior. Power law distributions are often used to model random walks, as they can characterize a wide range of behaviors, including Lévy walks. Empirical evidence indicates the prevalence and efficiency of Lévy walks as a foraging strategy, and theoretical work suggests an evolutionary origin. However, previous evolutionary models have assumed a priori that move lengths are drawn from a power law or other families of distributions. Here, we remove this restriction with a model that allows for the evolution of any distribution. Instead of Lévy walks, our model unfailingly results in the evolution of intermittent search, a random walk composed of two disjoint modes-frequent localized walks and infrequent extensive moves-that consistently outcompeted Lévy walks. We also demonstrate that foraging using intermittent search may resemble a Lévy walk because of interactions with the resources within an environment. These extrinsically generated Lévy-like walks belie an underlying behavior and may explain the prevalence of Lévy walks reported in the literature.

Multi-gene measurable residual disease assessed by digital polymerase chain reaction has clinical and biological utility in acute myeloid leukemia patients receiving venetoclax/azacitidine.

Venetoclax with azacitidine (ven/aza) is a lower-intensity therapeutic regimen that has been shown to improve outcomes in elderly patients with acute myeloid leukemia (AML). Measurable residual disease (MRD) using flow cytometry is a valuable tool for the prediction of relapse in AML using conventional therapies and ven/aza; however, the prognostic value for broadscale molecular MRD after ven/aza treatment is less clear. We aimed to determine the utility of retrospective assessment using multi-gene molecular MRD by droplet digital polymerase chain reaction (ddPCR). We found this approach correlates with outcomes in a cohort of patients receiving frontline ven/aza for AML. The predictive value of ddPCR MRD persisted when NPM1 mutations were removed from analysis, as well as after adjustment for the impact of stem cell transplant on outcomes. Late achievement of MRD negativity, including after SCT, was still associated with superior outcomes compared to persistently detectable MRD. We further explored the impact of ven/aza on the burden of different classes of mutations, and identified the persistence of splicing factor mutations, commonly associated with MDS, as a consistent finding after ven/aza treatment. These data add to our understanding of the effects of ven/aza on AML disease biology and provide details on molecular depth of remission that can guide prospective trials in the future.

CD46-ADC Reduces the Engraftment of Multiple Myeloma Patient-Derived Xenografts.

An antibody-drug conjugate (ADC) targeting CD46 conjugated to monomethyl auristatin has a potent anti-myeloma effect in cell lines in vitro and in vivo, and patient samples treated ex vivo. Here, we tested if CD46-ADC may have the potential to target MM-initiating cells (MM-ICs). CD46 expression was measured on primary MM cells with a stem-like phenotype. A patient-derived xenograft (PDX) model was implemented utilizing implanted fetal bone fragments to provide a humanized microenvironment. Engraftment was monitored via serum human light chain ELISA, and at sacrifice via bone marrow and bone fragment flow cytometry. We then tested MM regeneration in PDX by treating mice with CD46-ADC or the nonbinding control-ADC. MM progenitor cells from patients that exhibit high aldehyde dehydrogenase activity also have a high expression of CD46. In PDX, newly diagnosed MM patient samples engrafted significantly more compared to relapsed/refractory samples. In mice transplanted with newly diagnosed samples, CD46-ADC treatment showed significantly decreased engraftment compared to control-ADC treatment. Our data further support the targeting of CD46 in MM. To our knowledge, this is the first study to show preclinical drug efficacy in a PDX model of MM. This is an important area for future study, as patient samples but not cell lines accurately represent intratumoral heterogeneity.

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

We previously reported that acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL2, creating a therapeutic opportunity to target LSCs using the BCL2 inhibitor drug venetoclax. While venetoclax-based regimens have indeed shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence mechanisms that dictate venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e., OXPHOS) status with relatively high steady-state levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake sharply reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in the biology of LSCs and provide a therapeutic avenue for clinical management of venetoclax resistance.

LoCoMoTe - a Framework for Classification of Natural Locomotion in VR by Task, Technique and Modality.

Virtual reality (VR) research has provided overviews of locomotion techniques, how they work, their strengths and overall user experience. Considerable research has investigated new methodologies, particularly machine learning to develop redirection algorithms. To best support the development of redirection algorithms through machine learning, we must understand how best to replicate human navigation and behaviour in VR, which can be supported by the accumulation of results produced through live-user experiments. However, it can be difficult to identify, select and compare relevant research without a pre-existing framework in an ever-growing research field. Therefore, this work aimed to facilitate the ongoing structuring and comparison of the VR-based natural walking literature by providing a standardised framework for researchers to utilise. We applied thematic analysis to study methodology descriptions from 140 VR-based papers that contained live-user experiments. From this analysis, we developed the LoCoMoTe framework with three themes: navigational decisions, technique implementation, and modalities. The LoCoMoTe framework provides a standardised approach to structuring and comparing experimental conditions. The framework should be continually updated to categorise and systematise knowledge and aid in identifying research gaps and discussions.

A Novel Type of Monocytic Leukemia Stem Cell Revealed by the Clinical Use of Venetoclax-Based Therapy.

The BCL2 inhibitor venetoclax has recently emerged as an important component of acute myeloid leukemia (AML) therapy. Notably, use of this agent has revealed a previously unrecognized form of pathogenesis characterized by monocytic disease progression. We demonstrate that this form of disease arises from a fundamentally different type of leukemia stem cell (LSC), which we designate as monocytic LSC (m-LSC), that is developmentally and clinically distinct from the more well-described primitive LSC (p-LSC). The m-LSC is distinguished by a unique immunophenotype (CD34-, CD4+, CD11b-, CD14-, CD36-), unique transcriptional state, reliance on purine metabolism, and selective sensitivity to cladribine. Critically, in some instances, m-LSC and p-LSC subtypes can co-reside in the same patient with AML and simultaneously contribute to overall tumor biology. Thus, our findings demonstrate that LSC heterogeneity has direct clinical significance and highlight the need to distinguish and target m-LSCs as a means to improve clinical outcomes with venetoclax-based regimens. SIGNIFICANCE: These studies identify and characterize a new type of human acute myeloid LSC that is responsible for monocytic disease progression in patients with AML treated with venetoclax-based regimens. Our studies describe the phenotype, molecular properties, and drug sensitivities of this unique LSC subclass. This article is featured in Selected Articles from This Issue, p. 1949.

Therapy-Resistant Acute Myeloid Leukemia Stem Cells Are Resensitized to Venetoclax + Azacitidine by Targeting Fatty Acid Desaturases 1 and 2.

Recent advances in targeting leukemic stem cells (LSCs) using venetoclax with azacitidine (ven + aza) has significantly improved outcomes for de novo acute myeloid leukemia (AML) patients. However, patients who relapse after traditional chemotherapy are often venetoclax-resistant and exhibit poor clinical outcomes. We previously described that fatty acid metabolism drives oxidative phosphorylation (OXPHOS) and acts as a mechanism of LSC survival in relapsed/refractory AML. Here, we report that chemotherapy-relapsed primary AML displays aberrant fatty acid and lipid metabolism, as well as increased fatty acid desaturation through the activity of fatty acid desaturases 1 and 2, and that fatty acid desaturases function as a mechanism of recycling NAD+ to drive relapsed LSC survival. When combined with ven + aza, the genetic and pharmacologic inhibition of fatty acid desaturation results in decreased primary AML viability in relapsed AML. This study includes the largest lipidomic profile of LSC-enriched primary AML patient cells to date and indicates that inhibition of fatty acid desaturation is a promising therapeutic target for relapsed AML.

Higher-dose venetoclax with measurable residual disease-guided azacitidine discontinuation in newly diagnosed acute myeloid leukemia.

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.

Machine Learning-Based Exploratory Clinical Decision Support for Newly Diagnosed Patients With Acute Myeloid Leukemia Treated With 7 + 3 Type Chemotherapy or Venetoclax/Azacitidine.

PURPOSE: There are currently limited objective criteria to help assist physicians in determining whether an individual patient with acute myeloid leukemia (AML) is likely to do better with induction with either standard 7 + 3 chemotherapy or targeted therapy with venetoclax plus azacitidine. The study goal was to address this need by developing exploratory clinical decision support methods. PATIENTS AND METHODS: Univariable and multivariable analysis as well as comparison of a range of machine learning (ML) predictors were performed using cohorts of 120 newly diagnosed 7 + 3-treated AML patients compared with 101 venetoclax plus azacitidine-treated patients. RESULTS: A variety of features in the two patient cohorts were identified that may potentially correlate with short- and long-term outcomes, toxicities, and other considerations. A subset of these diagnostic features was then used to develop ML-based predictors with relatively high areas under the curve of short- and long-term outcomes, hospital stays, transfusion requirements, and toxicities for individual patients treated with either venetoclax/azacitidine or 7 + 3. CONCLUSION: Potential ML-based approaches to clinical decision support to help guide individual patients with newly diagnosed AML to either 7 + 3 or venetoclax plus azacitidine induction therapy were identified. Larger cohorts with separate test and validation studies are necessary to confirm these initial findings.

The evolutionary maintenance of Lévy flight foraging.

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.

Virtual Reality as a Pain Distraction Modality for Experimentally Induced Pain in a Chronic Pain Population: An Exploratory Study.

Virtual reality (VR) has shown promising results as an adjunct therapy for pain management. Recent literature exploring the use of VR for pain management among a chronic pain (CP) population has produced encouraging results, although little has been done to explore what about a VR intervention is the provider of the analgesic response. Furthermore, as has been suggested in the literature previously, little has been said of the association between pain tolerance and presence. This study primarily aimed to investigate pain tolerance differentiation between VR-head-mounted display (HMD) active and control interventions. Secondarily, this study looked to report on whether presence correlates to pain tolerance, among a CP population. A repeated-measures study design was used. Twelve participants received two 5-minute interventions while being subjected to experimentally induced pain. The interventions were as follows: (a) "active intervention," an immersive and interactive experience (b) "control intervention," and a nonimmersive controlled experience with no interaction. Tolerance to pain was assessed via the total time the participant continued the intervention. Presence was assessed via the Witmer and Singer's presence questionnaire. Participants also completed the Simulator Sickness Questionnaire, the Presence Questionnaire, and the Brief Pain Inventory. Pain tolerance was significantly higher in the active intervention compared with the control intervention (p = 0.005). There was a positive correlation between pain tolerance and presence during the active VR intervention. The media as opposed to the medium was determined to be responsible for greater tolerance to pain, as well as greater sense of presence, which was positively correlated to an increase in pain tolerance.

The STAT3-MYC axis promotes survival of leukemia stem cells by regulating SLC1A5 and oxidative phosphorylation.

Acute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.

Venetoclax and azacitidine compared with induction chemotherapy for newly diagnosed patients with acute myeloid leukemia.

Venetoclax (ven) plus azacitidine (aza) is the standard of care for patients with newly diagnosed acute myeloid leukemia (AML) who are not candidates for intensive chemotherapy (IC). Some patients who are IC candidates instead receive ven/aza. We retrospectively analyzed patients with newly diagnosed AML who received ven/aza (n = 143) or IC (n = 149) to compare outcomes, seek variables that could predict response to 1 therapy or the other, and ascertain whether treatment recommendations could be refined. The response rates were 76.9% for ven/aza and 70.5% for IC. The median overall survival (OS) was 884 days for IC compared with 483 days for ven/aza (P = .0020). A propensity-matched cohort was used to compare outcomes in the setting of equivalent baseline variables, and when matched for age, biological risk, and transplantation, the median OS was 705 days for IC compared with not reached for ven/aza (P = .0667). Variables that favored response to ven/aza over IC included older age, secondary AML, and RUNX1 mutations. AML M5 favored response to IC over ven/aza. In the propensity-matched cohort analyzing OS, older age, adverse risk, and RUNX1 mutations favored ven/aza over IC, whereas intermediate risk favored IC over ven/aza. In conclusion, patients receiving IC have improved OS compared with those receiving ven/aza. However, in a propensity-matched cohort of patients with equivalent baseline factors, there was a trend toward favorable OS for ven/aza. Specific variables, such as RUNX1 mutations, reported here for the first time, can be identified that favor ven/aza or IC, helping to guide treatment decisions for patients who may be eligible candidates for either therapy.

PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

Enriching for human acute myeloid leukemia stem cells using reactive oxygen species-based cell sorting.

Isolation of leukemia stem cells presents a challenge due to the heterogeneity of the immunophenotypic markers commonly used to identify blood stem cells. Several studies have reported that relative levels of reactive oxygen species (ROS) can be used to enrich for stem cell populations, suggesting a potential alternative to surface antigen-based methods. Here, we describe a protocol to enrich for stem cells from human acute myeloid leukemia specimens using relative levels of ROS. This protocol provides consistent enrichment of leukemia stem cells. For complete details on the use and execution of this protocol, please refer to Lagadinou et al. (2013) and Pei et al. (2018).

MDS-associated SF3B1 mutations enhance proinflammatory gene expression in patient blast cells.

Two factors known to contribute to the development of myelodysplastic syndrome (MDS) and other blood cancers are (i) somatically acquired mutations in components of the spliceosome and (ii) increased inflammation. Spliceosome genes, including SF3B1, are mutated at high frequency in MDS and other blood cancers; these mutations are thought to be neomorphic or gain-of-function mutations that drive disease pathogenesis. Likewise, increased inflammation is thought to contribute to MDS pathogenesis; inflammatory cytokines are strongly elevated in these patients, with higher levels correlating with worsened patient outcome. In the current study, we used RNAseq to analyze pre-mRNA splicing and gene expression changes present in blast cells isolated from MDS patients with or without SF3B1 mutations. We determined that SF3B1 mutations lead to enhanced proinflammatory gene expression in these cells. Thus, these studies suggest that SF3B1 mutations could contribute to MDS pathogenesis by enhancing the proinflammatory milieu in these patients.