Advances in multiagent chemotherapy have led to recent improvements in overall survival for patients with acute lymphoblastic leukemia (ALL); however, a significant fraction do not respond to frontline chemotherapy or later relapse with recurrent disease, after which long-term survival rates remain low. To address the challenge of developing new, effective treatment options for these patients, we conducted a series of high-throughput combination drug screens to identify chemotherapies that synergize in a lineage-specific manner with MRX-2843, a small molecule dual MERTK and FLT3 kinase inhibitor currently in clinical testing for treatment of relapsed/refractory leukemias and solid tumors. Using experimental and computational approaches, we found that MRX-2843 synergized strongly - and in a ratio-dependent manner - with vincristine chemotherapy to inhibit T-ALL cell expansion and, based on these findings, we developed multiagent lipid nanoparticle formulations of these drugs that not only constitutively maintained ratiometric drug synergy following T-ALL cell delivery, but also improved anti-leukemic activity following drug encapsulation. To determine the clinical relevance of these combination drug formulations and the therapeutic impact of ratiometric drug synergy, we compared the efficacy of lipid nanoparticles comprising synergistic, additive, and antagonistic ratios of MRX-2843 and vincristine, and observed that trends in in vitro synergy were directly recapitulated in primary T-ALL patient samples. Together, these findings present a systematic approach to high-throughput combination drug screening and multiagent drug delivery that maximizes the therapeutic potential of combined MRX-2843 and vincristine in T-ALL. This broadly generalizable approach could lead to the development of constitutively synergistic combination products for the treatment of cancer and other diseases.

No abstract

Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count greater than 100,000/μL, is an emergency in acute leukemia, possibly resulting in life-threatening microvascular obstruction, or leukostasis, leading to neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) complications. The underlying mechanisms remain poorly understood and are canonically attributed to blood hyperviscosity secondary to high WBC count and abnormal biophysical properties of leukemia cells themselves (leukemia immunophenotype, increased cell size, adhesion, and stiffness). Leukapheresis is a commonly-used therapy for rapid cytoreduction in symptomatic patients, but the procedure carries risk and existing guidelines are supported by scant evidence. Interestingly, despite hematocrit(Hct)/hemoglobin(Hgb) levels being major drivers of blood viscosity due to the high prevalence of circulating red cells (RBCs), how Hct/Hgb mediates hyperviscosity in acute leukemia is unknown. This is clinically important as Hct/Hgb often decrease as leukemic cell counts rise, and acute leukemia patients with anemia are often transfused. While sickle cell disease guidelines advise using a target post transfusion Hct of 30% to minimize iatrogenic hyperviscosity and its morbid complications, no guidelines have been established for acute leukemia. As such, can RBC transfusion actually increase leukostasis risk in acute leukemia? To explore this question requires new biophysical tools as the complexity of blood viscosity increases substantially at the microvascular level as the physical properties of the cells themselves become the major determinants of resistance to blood flow. To that end, we developed "microvasculature-on-a-chip" devices that recapitulate microvascular biophysical and hemodynamic conditions to investigate how the differing presentations of acute leukemia and transfusion support affect the effective blood viscosity at the microvascular level to cause "in vitro leukostasis." A multiple-vessel "multiplex" microfluidic device that operates at the appropriate size scale and mimics the microvascular geometry was designed to enable assessing accurate biophysical measurements of blood hyperviscosity. The devices were microfabricated using standard polydimethylsiloxane-based photolithography (Figure 1). Acute B-cell lymphoblastic (B-ALL, 697), acute T-cell lymphoblastic (T-ALL, Jurkat) and acute myelocytic (AML, HL60) leukemia cell lines were maintained via standard cell culture conditions. Patient samples were obtained through our institution's IRB. RBCs from healthy donors were isolated and mixed with leukemia cells to achieve target Hct/Hgb and WBC levels. Various physiologic leukemia "mixtures" were then perfused under physiologic microcirculatory flow conditions through the microvascular device and microchannels occlusion was tracked via videomicroscopy (Figure 2). Using a standard least squares multivariable linear regression with first and second order effects, microchannel size, Hct/Hgb, WBC count and leukemia cell type all showed statically significant effect on in vitro leukostasis, or microchannel occlusion over time, (all p values < 0.03) (Figure 3). Overall, severe anemia appears to be protective against in vitro leukostasis and there appears to be Hct/Hgb thresholds above which in vitro leukostasis becomes more prevalent, though this is different for B-ALL versus T-ALL. This is in contrast to AML, where severe anemia does not appear to offer protection against in vitro leukostasis as occlusion was appreciated at all Hct/Hgb levels. These data suggest when determining risk for leukostasis, WBC count and leukemia immunophenotype should not be the sole determinants. Here we show Hct/Hgb levels affect microvascular blood viscosity and risk for microvascular occlusion. These results may impact decisions regarding RBC transfusions and possibly initiation of leukapheresis in asymptomatic patients. Having a model to assess risk associated with RBC transfusions and informing clinicians when a patient might become at risk for leukostasis can have a significant impact on their clinical outcome, morbidity and mortality. Ongoing studies incorporating patient lymphoid and myeloid leukemia cells are needed to support this cell line data. Figure 1 Figure 1. Disclosures Kemp: Parthenon Therapeutics: Membership on an entity's Board of Directors or advisory committees. Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company.

Background: Sickle cell disease (SCD) is a group of genetic disorders in which sickle hemoglobin polymerizes under deoxygenation, altering red blood cell (RBC) morphology and behavior. The properties of sickle RBCs contribute to increased viscosity of blood and occurrence of vaso-occlusions, a major aspect of SCD pathophysiology. Voxelotor is a novel FDA-approved treatment for SCD which modulates hemoglobin O 2 affinity, and while its known mechanism inhibits sickle polymerization, the impact on other aspects of SCD pathophysiology remain unknown. Thus, despite the new treatment option, highly variable clinical manifestation continues to be a hallmark of sickle cell and there is consequently a need to optimize the use of current therapies based on patient-specific factors. In this work, we leverage datasets generated from a unique microfluidic assay that measures blood flow behavior under varying oxygen tension in conjunction with novel statistical approaches to model and assess sources of variability in sickle blood flow response to voxelotor. Methods: RBCs from patients with SCD (n=28) were treated with voxelotor at 500 uM concentration. Treated samples and untreated controls were perfused through a microfluidic platform that dynamically modulates oxygen tension and measures flow velocity (Wood et al, 2012; Valdez et al, 2019). The area between curves (ABC) of the normalized velocity across the range of oxygen tension between treated and control conditions was calculated to quantify the effect of voxelotor for each sample (figure 1). A paired t-test was used to assess the difference in response between treated and untreated samples. Where available, clinical data including the hemoglobin fractions and complete blood count (CBC) were collected for each sample as predictor variables, and partial least squares regression (PLSR) modeling was used to assess the correlation of predictors and responses. Results: Voxelotor increased the velocity ABC from untreated to treated conditions (p<.0001). We observed that there were differences in response for velocity ABC between sickle cell genotypes (figure 2). Thus, generating separate PLSR models for distinct SCD genotypes revealed differences in sets of clinical factors that explained the most variance in response to voxelotor treatment. A 2-component model was constructed for the HbSC samples (n=6) that best explained variance in the data and had good predictive abilities (R 2X=.69, R 2Y=.97, Q 2=.74). Within this subset, clustering of variables related to hemolysis and inflammation were observed (figure 3). An equivalent model constructed for the HbSS samples (n=15) characterized the predictor variables but lacked predictive power of the response (R 2X=.74, R 2Y=.25, Q 2=.-0.21). Response to voxelotor for this model was most strongly correlated with HbA. Due to low sample size (n=2 samples with full set of predictors), predictive modeling was not performed for HbSβ 0 samples, however, these samples responded the least to voxelotor treatment. Conclusions: Our analysis quantified patient-specific differences in the blood flow response to voxelotor, showing a wide variability in response despite treatment by the same drug concentration. Genotype-specific multivariable models that take into account easily measurable clinical variables such as the CBC have the potential to explain the variability in patient response to voxelotor treatment. In HbSC samples, the WBC, platelet, and reticulocyte counts were highly correlated and strong predictors of response to voxelotor, which may point to markers of hemolysis and inflammation being useful in determining patients that can be optimally treated with this drug. In HbSS, response to voxelotor was mainly inversely correlated with HbA levels, which is a surrogate marker for blood transfusions, indicating that the effect of voxelotor is lessened for patients who are receiving transfusions. However, the low R2Y of this model highlights the clinical variability in this SCD genotype and consequent need for additional biomarkers of disease severity. In conclusion, our hybrid experimental-computational approach is able to identify clinical factors that highly impact the response of patient blood samples to treatment with voxelotor for HbSC patients, and highlights the need for precision therapy recommendations in SCD. Figure 1 Figure 1. Disclosures Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company. Kemp: Parthenon Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Red cell transfusions are an effective part of a clinical care regiment in the treatment of chronic sickle cell disease; however, the understanding of the target hemoglobin levels has not been investigated past the standard hematocrit/hemoglobin (HgB) of 10 g/dL. A simple transfusion of packed red cells can be a beneficial clinical treatment of acute pain crisis or even stroke. Along with other transfusion-based complications, when performing a simple transfusion, the changes in blood velocity as a result of increased blood viscosity from the additional red cells can lead to complications of their own. Because of this, clinical treatment has hesitated to transfuse sickle patients above a HgB of 10 g/dL. The complications of sickle cell disease tend to be more pronounced on the microvascular scale than then macrovascular. Along with this, the overall slower blood flow caused by the increase in viscosity from a simple blood transfusion is more probable to lead to complications on the microvascular level. Our device allows us to target the changes in whole blood on multiple scales including down to arteriole sizes. Here, we have begun to investigate how transfusion could be more patient-specific by identifying the velocity profile of whole blood flowing through a "microvasculature-on-a-chip" device that mimics the microvascular geometry (Figure 1A). The devices were microfabricated using polydimethylsiloxane (PDMS) and then coated with 0.1% bovine serum albumin (BSA) to help prevent red cell adhesion to channel walls. To simulate various HgB levels, healthy whole blood samples were centrifuged to separate red cells. To simulate a simple clinical transfusion of a sickle patient, isolated red cells were added to sickle whole blood samples. Similar to a clinical setting, sickle samples were only transfused up to higher HgB levels. HgB levels were then confirmed on a differential hematology analyzer (Sysmex XN 330). 3.2mm CA+ was added to various HgB samples to defeat the citrate anticoagulant. Samples were loaded into syringes then perfused into the BSA coated devices (Figure 1B). During perfusion, a 450 frame video of flow was captured at 40x resolution and 163 fps. Following capture, videos parameters such as frame rate and pixel distance were defined in a custom MATLAB (Mathworks, Natick, MA) script. The script segmented videos into cropped frames of the desired regions of interest then a Kanade-Lucas-Tomasi (KLT) tracking algorithm detected red cell features in each frame across 4 frames (Figure 1 B&C). 12 equal spaced bins were created across the width of the channel in the direction of flow; Tracked velocities were assigned to their corresponding bin and averaged to create a velocity profile of function as the distance from the center of the channel (Figure 1 D&E). To create a case study, two patient samples were received with the same starting HgB of 6.8 g/dL and were transfused upwards incrementally to a HgB of 12 g/dL. One patient is currently on a hydroxyurea regiment and the other patient is not. At each HgB level, the perfused whole blood was tracked through several different arteriole-sized vessels (30, 40 & 60 um) at two appropriate flow rates. To quantify the differences in the flow, the average cell velocity (um/s) through the channel and the peak velocity (um/s) through channels were charted against the various HgB levels (Figure 2). Continuing this series of experiments, 2 additional sickle whole blood on hydroxyurea samples were transfused upwards from their respective starting hemoglobin (9.7 & 10 g/dL). The flow was tracked and averages were quantified across the channel through its distance from the center of the channel. As transfused sickle HgB levels were increased, the bluntness of the velocity profile, or the difference between the average flow velocity in the center of the channel and at the walls of the channel, became less dramatic. This could be primarily attributed to the increase in the viscosity from the addition of the red cells (Figure 3). Our data shows that viscosity plays an important factor in whole blood flow. HgB of 10 g/dL is an important target for sickle transfusions; however, this target HgB may be more patient-specific than previously stated. Understanding patient viscosity may prove to be more important than hemoglobin levels. As patient blood increases in viscosity, blood slows down on the microvascular level the most. This may be critical in understanding the appropriate transfusion. Figure 1 Figure 1. Disclosures Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company. Kemp: Parthenon Therapeutics: Membership on an entity's Board of Directors or advisory committees.