Myeloid Targeted Human MLL-ENL and MLL-AF9 Induces cdk9 and bcl2 Expression in Zebrafish Embryos

Acute myeloid leukemia (AML) accounts for greater than twenty thousand new cases of leukemia annually in the United States. The average five-year survival rate is approximately 30%, pointing to the need for developing novel model systems for drug discovery. In particular, patients with chromosomal rearrangements in the mixed lineage leukemia (MLL) gene have higher relapse rates with poor outcomes. In this study we investigated the expression of human MLL-ENL and MLL-AF9 in the myeloid lineage of zebrafish embryos. We observed an expansion of MLL positive cells and determined these cells colocalized with the myeloid markers spi1b, mpx, and mpeg. In addition, expression of MLL-ENL and MLL-AF9 induced the expression of endogenous bcl2 and cdk9, genes that are often dysregulated in MLL-r-AML. Co-treatment of lyz: MLL-ENL or lyz:MLL-AF9 expressing embryos with the BCL2 inhibitor, Venetoclax, and the CDK9 inhibitor, Flavopiridol, significantly reduced the number of MLL positive cells compared to embryos treated with vehicle or either drug alone. In addition, cotreatment with Venetoclax and Flavopiridol significantly reduced the expression of endogenous mcl1a compared to vehicle, consistent with AML. This new model of MLL-r-AML provides a novel tool to understand the molecular mechanisms underlying disease progression and a platform for drug discovery.


Introduction
Acute myeloid leukemia (AML) is characterized by an accumulation of hematopoietic precursor cells in the bone marrow and blood as a result of multiple gene mutations and acquisition of chromosomal rearrangements [1].In AML, mixed lineage leukemia (MLL1 or KMT2A) frequently undergoes chromosomal rearrangements resulting in the production of an oncogenic fusion protein comprised of the N-terminus of MLL and the C-terminal portion of a fusion partner [2,3].Although many MLL rearrangements (MLL-r) have been identified, approximately half of MLL-r-AML patients harbor a genetic fusion with AF4, AF9, AF10, or ENL [3,4].These proteins are important components of the AF4 super elongation complex (SEC) which also includes the positive transcription elongation factor b (p-TEFb) complex, bromodomain containing 4 (BRD4), and DOT1 like histone lysine methyltransferase (DOT1L).Ectopic expression of these fusion proteins leads to aberrant histone 3 lysine 79 (H3K79) methylation via DOT1L, as well as increased transcriptional elongation through p-TEFb.[5].Patients harboring an MLL-r have a higher relapse rate and their overall survival is low [3].Therefore, new therapeutic treatment protocols need to be investigated for patients with MLL-r AML.
In this study, expression of human MLL-ENL and MLL-AF9 in the myeloid lineage resulted in expansion and clustering of MLL positive cells on the yolk of zebrafish embryos.Marker analysis confirmed colocalization of MLL with myeloid markers sp1b, mpx, and mpeg but not lyz.Further examination identified increased expression of endogenous cdk9 and bcl2 on the yolk of MLL-ENL and MLL-AF9 expressing embryos but not in controls.Coincubation of MLL-ENL or MLL-AF9 expressing embryos with Venetoclax, a BCL2 inhibitor, and Flavopiridol, a CDK9 inhibitor, resulted in significantly fewer MLL-ENL positive cells than treatment with vehicle or either drug alone.Therefore, this study demonstrates that expression of MLL-ENL or MLL-AF9 in the myeloid lineage induced an expansion of myeloid lineage cells as well as endogenous cdk9 and bcl2 expression in zebrafish embryos, and that cooperative inhibition of Bcl2 and Cdk9 is an effective treatment for MLL-r myeloproliferation.

Myeloid expression of MLL-ENL in zebrafish embryos
Zebrafish have emerged as a powerful model system to study leukemogenesis by targeting known human fusion proteins in the myeloid or lymphoid lineage [21].In particular, the fusion of the MLL (Lysine [K]-specific MethylTransferase 2A) gene to either ENL (MLLT1, Myeloid/Lymphoid or Mixed-Lineage Leukemia) or AF9 (MLLT3, Myeloid/Lymphoid or Mixed-Lineage Leukemia) are common rearrangements found in AML patients [3].To determine if MLL-ENL or MLL-AF9 fusion proteins alter myeloid development in zebrafish, human FLAG-tagged MLL-ENL or FLAG-tagged MLL-AF9 were expressed under the zebrafish lysozyme C (lyz) promoter [27][28][29].To confirm insertion of the construct into the genome, the final expression vector included the α-crystallin promoter driving EGFP expression in the lens (Fig 1A -1C) [30].Similar to expression of other human fusion proteins, stable transgenic fish expressing lyz:MLL-ENL or lyz:MLL-AF9 in the myeloid lineage did not survive, likely due to altered myelopoiesis inhibiting maturation [10].Therefore, all analyses for this research were completed using transient transgenic embryos.Lyz is expressed in myeloid progenitor cells, neutrophils, and macrophages during zebrafish development.Its expression is first detected on the yolk at 22 hours post fertilization (hpf), where it colocalizes with the myeloid progenitor marker, spi1b, on the yolk and in the intermediate cell mass (ICM) (S1 Fig) [27,29].At 25 hpf, lyz positive cells migrate ventrally to the duct of Cuvier and enter into circulation to seed the peripheral blood island (PBI)/caudal hematopoietic tissue (CHT) [27].Therefore, expression of lyz:MLL-ENL and lyz:MLL-AF9 were analyzed at 30 hpf, 48 hpf, 72 hpf, and 7 days post fertilization (dpf) using a probe that detects human MLL (MLL).Consistent with lyz expression in developing embryos, expression of MLL was observed on the yolk at 30 hpf (Fig 1E and 1F).Further analysis of lyz:MLL-ENL and lyz: MLL-AF9 injected embryos at 48 hpf, 72 hpf, and 7 dpf identified expression of MLL exclusively on the yolk, often in clusters (Fig 1E , 1F, 1H, 1I, 1K, 1L, 1N and 1O).The absence of expression in uninjected embryos confirmed that the MLL probe did not cross react with endogenous zebrafish mll (Fig 1D , 1G, 1J and 1M).Given lyz is expressed in the ICM and MLL expression was observed exclusively on the yolk, we further validated the specificity of the lyz promoter.We expressed mCherry under the lyz promoter and identified mCherry+ cells in the CHT at 72 hpf (S2 Fig), confirming promoter specificity.Therefore, expression of MLL-ENL or MLL-AF9 in the myeloid lineage induced the expansion of MLL expressing cells on the yolk of developing zebrafish embryos.

Accumulation of MLL positive cells on the yolk is not due to cardiovascular defects
MLL positive cells were exclusively found on the yolk in lyz:MLL-ENL expressing embryos, which is in contrast to wild type embryos, where lyz positive cells enter into circulation to seed the PBI/CHT [27].To determine if MLL-positive cells remained on the yolk due to defects in cardiovascular development or blood flow, we analyzed heart rate, blood circulation, and endogenous lyz expression in lyz:MLL-ENL injected embryos.The heart rate was significantly, but not severely, decreased (133 bpm +/-5.6)compared to uninjected control embryos (157 +/-9.5) at 72 hpf (Fig 2A).However, this decrease in heart rate did not inhibit circulation of red blood cells at 48 hpf (S1 and S2 Videos) or spi1b:GFP positive cells at 72 hpf (S3 and S4 Videos) when examined in confirmed MLL-ENL expressing embryos.In addition, circulation was also confirmed by the expression of endogenous lyz positive cells in the CHT of lyz:MLL-ENL expressing embryos at 72 hpf (Fig 2B -2E), where the number of lyz positive cells was similar to uninjected controls.This result suggests that lyz positive cells that did not express MLL-ENL were able to seed the CHT (Fig 2F).Furthermore, we analyzed hematopoietic stem and progenitor cell (HSPC) markers, tal1 and runx1/cmyb, in MLL-ENL and MLL-AF9 expressing embryos since blood flow and myeloid cell signaling are necessary for HSPC specification [31,32].Tal1 and runx1/cmyb expression were normal in confirmed lyz:MLL-ENL and lyz:MLL-AF9 expressing embryos compared to uninjected controls (Fig 2G -2L).
To better understand why MLL positive cells remained on the yolk, we employed timelapse imaging of spi1b:GFP embryos injected with lyz:MLL-AF9.Control spi1b:GFP positive cells were observed forming protrusions and rapidly migrating over the yolk at 48hpf (Fig 2M and S5 Video).In contrast, a subset of spi1b:GFP positive cells formed short protrusions and did not migrate but remained in the same location on the yolk in embryos injected with lyz: MLL-AF9 (Fig 2N and S6 Video).Therefore, the accumulation of MLL-ENL and MLL-AF9 expressing cells on the yolk likely resulted from impaired migration and was not a result of defects in cardiovascular development.
To confirm the results of single marker gene analyses (Figs 3I and 4I), double colorimetric whole mount in situ hybridization was completed on lyz targeted MLL-ENL and MLL-AF9 expressing cells.The total number of stained cells on the yolk was counted and the percentage of cells that expressed the marker gene (lyz, mpx, spi1b, or mpeg), MLL, or both marker gene and MLL was calculated.Consistent with single probe analysis (Fig 3I ), MLL did not colocalize with lyz at 48 or 72 hpf (Figs 3J, 3N, 4J and 4N).The percentage of stained cells on the yolk that expressed endogenous lyz decreased from ~55% at 48 hpf to ~12% at 72 hpf, while the percentage of stained cells that expressed MLL increased from ~40% to over 90% at the same timepoints in both MLL-ENL and MLL-AF9 expressing embryos (Figs 3J, 3N, 4J and 4N).These results suggest that lyz expression is reduced or lost in cells that express MLL-ENL or MLL-AF9.
In addition to dysregulated transcription, inhibition of apoptosis through increased expression of pro-survival genes promotes cell survival and myeloid expansion in AML.One key gene that inhibits apoptosis in AML is B-cell lymphoma 2 (BCL2) [38,40,41].Therefore, ).Thus, expression of human MLL-ENL or MLL-AF9 in the myeloid lineage of zebrafish embryos leads to dramatically increased expression of endogenous cdk9 and bcl2, similar to mouse models and patients with MLL-r-AML [37,42].

Dose dependent effects of Venetoclax and Flavopiridol on myelopoiesis in zebrafish embryos
To assess the effects of dysregulated cdk9 and bcl2 expression on zebrafish embryos, pharmacological inhibitors of these two target proteins were investigated.The BCL2 inhibitor, Venetoclax, was selected because it is a small-molecule selective inhibitor that is currently approved to treat patients with chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), and newly diagnosed AML in combination with other therapies [43].Control embryos were treated with 100 and 500nM of Venetoclax beginning at 24 hpf and assessed for expression of myeloid markers (lyz, mpx, mpeg) and the myeloid and hematopoietic stem cell marker, cmyb (Fig 7A -7L).Expression of lyz and mpx were unchanged in embryos treated with 100nM of Venetoclax, but were significantly decreased at 500nM (Fig 7M).Hematopoietic stem cells in the CHT were assessed by cmyb expression at 48 hpf and found to be unaffected at 100nM and 500nM concentrations compared to control embryos (Fig 7M).Importantly, inhibition of Bcl2 at 100nM or 500nM did not cause any visible gross morphological defects at 48 or 72 hpf.Therefore, inhibition of Bcl2 using 500nM of Venetoclax inhibits myeloid but not hematopoietic development.
To determine the effects of Cdk9 inhibition on zebrafish development, a small-molecule inhibitor, Flavopiridol, was employed.Flavopiridol is a flavonoid alkaloid inhibitor that binds to the ATP-binding site of CDKs, reversibly inhibiting their function.It has been evaluated in clinical trials to treat AML in combination with other pharmacological inhibitors [44].Treatment of zebrafish embryos beginning at 24 hpf with 100nM or 500nM Flavopiridol did not cause any gross morphological defects at 48 or 72 hpf (Fig 7N -7Z).These concentrations are significantly less than those tested previously in zebrafish embryos, wherein 5μM caused edema and a curved body axis [45].Embryos incubated with 100nM Flavopiridol had similar numbers of lyz, mpx, mpeg, and cmyb positive cells in the CHT compared to control embryos.Treatment at 500nM caused a significant decrease in lyz positive cells, but did not affect mpx, mpeg, or cmyb positive cells in the CHT (Fig 7Z).Therefore, treatment of embryos with 500nM Flavopiridol reduced the number of lyz positive but not mpx positive myeloid cells or cmyb positive hematopoietic stem cells in the CHT.To further characterize the reduction of MLL positive cells in Venetoclax and Flavopiridol co-treated embryos, we analyzed levels of the proapoptotic gene, tp53, and the pro-survival gene, mcl1a.Treatment of lyz-MLL-ENL embryos with Venetoclax or lyz:MLL-AF9 embryos with Flavopiridol induced a significant reduction in endogenous tp53 expression compared to vehicle treatment alone, while the remaining treatment groups did not show a significant change in tp53.These results suggest that the reduction in MLL positive cells occurs in a Tp53-independent manner (Fig 8K and 8M).This is consistent with previous reports that demonstrate Venetoclax and Flavopiridol induced cell death independent of TP53 in leukemia cell lines [46,47].Mcl-1 is a member of the Bcl-2 family of prosurvival genes and is often overexpressed in AML [48,49].Venetoclax is thought to increase the binding affinity of Mcl-1 to proapoptotic proteins, such as Bim.Therefore, Venetoclax treatment alone is reported to have a limited effect on cancers that have elevated Mcl-1 [50].Flavopiridol inhibits pTEFb activation resulting in down-regulation of short-lived proteins, such as Mcl-1, in the absence of active transcription [51].Thus, Flavopiridol increases the sensitivity of Venetoclax in Mcl-1 elevated leukemia [38,49,52,53].Co-treatment with Flavopiridol and Venetoclax in lyz: MLL-ENL and lyz:MLL-AF9 expressing embryos significantly reduced the level of mcl1a, consistent with AML models.Therefore, inhibition of Bcl2 and Cdk9 reduces mcl1a expression leading to a partial recovery of the myeloid expansion observed in lyz:MLL-ENL and lyz: MLL-AF9 injected embryos.

Discussion
MLL rearrangements (MLL-r) occur in approximately 10% of AML patients, with 34% of those harboring an MLL-ENL or MLL-AF9 rearrangement.Despite modern therapies, patients with MLL-r-AML have poor outcomes compared to those with non-MLL-r-AML [54].Therefore, generating new animal models is critical to identify novel drug treatments to improve patient outcomes.Our results demonstrated that expression of MLL-ENL or MLL-AF9 in the myeloid lineage caused an expansion of myeloid cells and increased expression of endogenous cdk9 and bcl2 on the yolk of zebrafish embryos.MLL-ENL expressing embryos co-treated with the CDK9 inhibitor, Flavopiridol, and BCL2 inhibitor, Venetoclax, significantly reduced the number of MLL positive cells compared to vehicle and individual treatments alone.Such results are consistent with previous reports indicating that other CDK9 inhibitors, by inhibiting the pTEFb transcription elongation factor, can increase the anti-tumor effects of Venetoclax AML models [55].These results further support the use of zebrafish to recapitulate AML phenotypes and identify novel treatments for therapeutic intervention.
Stable lines of lyz:MLL-ENL and lyz:MLL-AF9 were sought using the Tol2-mediated transgenesis system [6] but all fish that were raised lacked stable germline insertion in the myeloid lineage.Our inability to raise stable lines could be a result of embryonic myeloid and hematopoietic deficiencies that resulted from MLL-r insertion.Therefore, all analysis for this research focused on transient transgenic embryos.
Primitive myeloid cells arise from the lateral plate mesoderm (LPM) derived rostral blood island (RBI) and the inner cell mass (ICM) during zebrafish development [11,12].Spi1b positive cells migrate to the anterior yolk and begin to express lyz at approximately 22 hpf.At the onset of blood flow, a subset of lyz positive cells then enter circulation to populate the ICM [11,[27][28][29]33].We identified single positive lyz and spi1b cells as well as double positive lyz and spi1b cells on the yolk, while all lyz positive cells in the ICM co-expressed spi1b in control embryos at 24 hpf [27,29].In our model, MLL expression was only found on the yolk and not identified in the ICM of lyz:MLL-ENL injected embryos.Furthermore, lyz positive cells that did not express MLL-ENL were able to enter circulation to populate the ICM.Timelapse imaging of spi1b:GFP positive cells in lyz:MLL-AF9 injected embryos suggested reduced migration inhibited these cells from entering circulation and induced the MLL expansion observed on the yolk in transgenic embryos.The absence of lyz colocalization with MLL was unexpected given the lyz promoter drove expression of both MLL-ENL and MLL-AF9.This result suggested that lyz expression was reduced or lost in these cells possibly pointing to a role for MLL-ENL and MLL-AF9 in regulating the transcription or stability of lyz transcripts.We also observed an increase in the number of spi1b and mpx positive cells on the yolk which was consistent with previous models [10,18,23,24,26].The increase in mpeg positive cells in lyz: MLL-ENL and lyz:MLL-AF9 expressing embryos suggested that these cells belong to the monocytic lineage.However, MLL differentially colocalized with spi1b, mpx, and mpeg markers in both MLL-ENL and MLL-AF9 expressing embryos and the presence of single positive MLL cells in all embryos examined suggested variable myeloid cell maturation.Our results are similar, but not identical, to previous research in zebrafish embryos where ectopically expressed MLL-AF9 mRNA increased expression of myeloid markers including lcp1, mpx, spi1b, and lyz, as well as the hematopoietic markers, runx1 and cmyb [26].Our results are likely different due (E,J).Lyz:MLL-ENL (K,L) and lyz:MLL-AF9 (M,N) injected embryos were assessed for expression of tp53 and mcl1a by qPCR (N = 2).Statistical analysis was conducted using one-way ANOVA followed by Tukey HSD.* p<0.05.Anterior to the left.https://doi.org/10.1371/journal.pgen.1011308.g008to targeted expression of MLL-ENL and MLL-AF9 to the myeloid lineage as opposed to ubiquitous expression using mRNA.
MLL-r-AML is characterized by dysregulated cell proliferation and reduced apoptosis.BCL2 is a key survival gene that is overexpressed in both AML patients and cell lines [42].BCL2 sequesters proapoptotic proteins, such as BIM, to inhibit cell death and arrest cells in G0 [56,57].We determined that expression of MLL-ENL or MLL-AF9 in myeloid cells resulted in increased expression of endogenous bcl2, similar to AML patients.Increased expression of bcl2 can be a consequence of dysregulated CDK9 which leads to altered transcriptional elongation and mRNA maturation of target genes, including MCL-1 [37].Zebrafish embryos expressing lyz:MLL-ENL and lyz:MLL-AF9 exhibited increased expression of cdk9 compared to controls.The observed increase in bcl2 and cdk9 was exclusively on the yolk, consistent with human MLL expression in MLL-ENL or MLL-AF9 expressing embryos.To determine if Bcl2 and Cdk9 inhibition alters normal embryonic development we employed Venetoclax (ABT-1099) and Flavopiridol, respectively.Venetoclax inhibits the interaction between BCL2 and BIM, allowing BIM to initiate apoptosis [42,43,56].Flavopiridol is a CDK family pharmacological inhibitor that preferentially targets CDK9 by binding to the ATP site inhibiting protein activity and preventing transcription of target genes such as MCL-1, which confers resistance to Venetoclax [38,44,58,59].We used low concentrations that were physiologically relevant to humans since previously it was shown that higher doses (1-5uM) of Flavopiridol caused morphological defects in zebrafish embryos, including tail curvature and edema [45].Wild type zebrafish embryos treated with low concentrations of Venetoclax and Flavopiridol were morphologically normal with only a significant reduction in lyz positive cells at 500nM of either drug and a significant reduction in mpx positive cells with 500nM Venetoclax.Similar to findings in AML cell lines, we observed a significant reduction in the number of MLL positive cells in MLL-ENL and MLL-AF9 expressing embryos co-incubated with Venetoclax and Flavopiridol compared to embryos treated with vehicle or either drug alone.In addition, co-treatment of MLL-ENL and MLL-AF9 embryos with Venetoclax and Flavopiridol significantly decreased endogenous mcl1a expression, consistent with AML patients and cell culture.Taken together, our results demonstrated that expression of human MLL-ENL or MLL-AF9 in the myeloid lineage of zebrafish embryos induced expression of bcl2 and cdk9, leading to myeloid cell expansion, similar to patients with MLL-r-AML.
Future research will seek to use additional pharmacological inhibitors to determine if synergistic treatments improve outcomes without negatively affecting endogenous myeloid cell development and maintenance.Thus, our results further support the use of zebrafish as a valuable preclinical model for hematological malignancies and will be advantageous in identifying novel targets for drug discovery.
MLL-ENL was cloned into the pME vector (Invitrogen) using EcoRI and MLL-AF9 was cloned into the pME vector using XhoI.Directionality was confirmed using sequencing.Gateway recombination technology (Invitrogen) was used to generate the lyz targeted MLL-ENL and MLL-AF9 vectors.The destination vector contained α-crystallin:EGFP which drives enhanced green fluorescent protein (EGFP) expression in the lens of zebrafish embryos beginning at 2 dpf.Embryos were injected at the one cell stage with 25 nanograms (ng) of lyz:MLL-ENL or lyz:MLL-AF9 and 25 ng of transposase mRNA, and raised under standard conditions.
Double colorimetric WISH was completed using fluorescein labeled MLL riboprobe that was synthesized as described above.Fluorescein-labeled MLL and digoxigenin-labeled mpx, spi1b, mpeg, lyz, tal1, runx1, and cmyb probes were hybridized to embryos and developed as previously described [68].Embryos were mounted in 3% methyl cellulose and imaged using a Nikon AZ100 multi-zoom fluorescent stereo microscope using Elements 4.50 software.

Venetoclax and Flavopiridol treatment
Embryos were dechorionated and resuspended in 1 mL of 1X E3 media with 0.004% 1-phenyl-2-thiourea (PTU) and incubated with Venetoclax or Flavopiridol (MedChem Express) at the desired drug concentration in 12-well dishes beginning at 24 hpf.Dimethyl sulfoxide (DMSO) was used to solubilize the drugs and was also used as a vehicle for control experiments.Drug treatments were continuous at 28˚C until 72 hpf when embryos were fixed in 4% PFA/PBS.
For recovery experiments, lyz:MLL-ENL injected embryos were dechorionated and split evenly in a 12 well dish.Embryos were then incubated in 1X E3 media and 0.004% PTU containing either 200 nM Venetoclax, 200 nM Flavopiridol, 200 nM Venetoclax and Flavopiridol, or DMSO beginning at 24 hpf at 28˚C before being fixed in 4% PFA/PBS at 72 hpf.
All reactions were performed in triplicate using 2x SensiFAST SYBR No-ROX Mix (Bioline, BIO-98005).qPCR analysis was performed using a micPCR system (Bio Molecular Systems, M0000636).Single product amplification was confirmed using both a melt curve and direct sequencing of the product.Gene expression fold change was analyzed using the 2 -ΔΔC T method and was normalized to elongation-factor 1-alpha (ef1α) [69].

Video microscopy
Live embryos were imaged using differential interference contrast optics or epifluorescence after transient anesthesia and immobilization on a Nikon AZ100 multi-zoom fluorescent stereo microscope using Elements 4.50 software.For each condition, embryos were imaged, individually fixed, and analyzed for MLL expression.5-10 embryos were analyzed per condition.Timelapse videos of Spi1b:GFP embryos were imaged every thirty seconds for ten minutes on a Nikon C2 laser scanning confocal on a Nikon Eclipse Ni microscope using Elements AR 4.50 software.

Statistical analysis
Statistical analyses were conducted using one-way ANOVA followed by Tukey's HSD for pharmacological inhibitor treatments and qPCR.Statistical analysis was conducted using Student's t-test for marker gene analysis and heart rates.Center values are calculated as the mean and error bars are standard deviations.

Fig 7 .
Fig 7. Dose dependent effect of Venetoclax and Flavopiridol on myelopoiesis.Embryos were treated with 100nM or 500nM of Venetoclax (A-M) or 100nM and 500nM Flavopiridol (N-Z) and assessed for expression of lyz (A-C, N-P, N = 10), mpx (D-F, Q-S, N = 16-19), mpeg (G-I, T-V, N = 20), and cmyb (J-L, W-Y, N = 16-20) by counting the number of positive cells expressing each marker in the CHT (M,Z).Arrows indicate marker gene expression in the CHT.Statistical analysis was conducted using one-way ANOVA followed by Tukey HSD.* p<0.05, ** p<0.01,NS = not significant.Anterior to the left.https://doi.org/10.1371/journal.pgen.1011308.g007 To determine if cdk9 and bcl2 promotes myeloid expansion in lyz:MLL-ENL and lyz:MLL-AF9 injected embryos, Venetoclax and Flavopiridol were investigated individually and in combination.Each drug was first assessed at 200nM in wild type embryos (S3 Fig) to determine its effect on myeloid development in control embryos.Treatment of embryos beginning at 24 hpf showed no significant change in the number of lyz positive cells in Venetoclax or combinatorially treated embryos, but did show a significant reduction for Flavopiridol alone when assessed at 72 hpf (S3 Fig).Furthermore, gross morphological defects were not observed in embryos treated with either drug alone or in combination.Therefore, 200nM Venetoclax or Flavopiridol alone or in combination were used in lyz:MLL-ENL and lyz:MLL-AF9 injected embryos.To assay rescue of myeloid expansion, MLL positive cells were counted on the yolk at 72 hpf.Vehicle treated MLL-ENL embryos averaged 61.4 (+/-56.4)and vehicle treated MLL-AF9 embryos averaged 35.1 (+/-29.3)MLL positive cells per embryo, while Venetoclax and Flavopiridol alone averaged 53.5 (+/-49.5)and 42.9 (+/-44.2) positive cells in MLL-ENL embryos and 30.3 (+/-25.7)and 27.9 (+/-28.9) in MLL-AF9 embryos, respectively (Fig 8A-8J).Neither drug treatment alone caused a statistically significant reduction in MLL expressing cells.However, treatment with a combination of Venetoclax and Flavopiridol significantly reduced the number of MLL positive cells on the yolk to 40.2 (+/-40.8) in lyz:MLL-ENL injected embryos and 24.7 (+/-21.5) in lyz:MLL-AF9 injected embryos (Fig 8E and 8J).Thus, combinatorial treatment of lyz:MLL-ENL and lyz:MLL-AF9 embryos partially recovered the myeloid expansion observed on the yolk.