Amino Acid Stress Response Genes Promote L-Asparaginase Resistance in Pediatric Acute Lymphoblastic Leukemia

Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcome. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood, beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP resistant and sensitive ALL cell lines (n = 7) as well as primary leukemia samples from newly diagnosed patients. We identified 2219 response genes (absolute log 2FC > 1.5, FDR p-value <0.05) with ~16.5% being reproduced in more than one cell line. Defining target genes of the amino acid stress response related transcription factor ATF4 in ALL cell lines using ChIP-seq revealed 25% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor. A total of 17,117 significantly differentially bound ATF4 sites were identified (FDR p-value <0.01) and 97.8% of these sites displayed an increase in ATF4 binding following LASP treatment. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells from 212 newly diagnosed children enrolled on St. Jude Total Therapy 16. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlights that many response genes may not differ in their basal expression in drug resistant leukemia cells. Disclosures Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Mullighan: Amgen: Current equity holder in publicly-traded company; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Pfizer: Research Funding. Pui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee. Evans: Princess Máxima Center for Pediatric Oncology, Scientific Advisory Board, Chair: Membership on an entity's Board of Directors or advisory committees; BioSkryb, Inc.: Membership on an entity's Board of Directors or advisory committees; St. Jude Children's Research Hospital, Emeritus Member (began Jan 2021): Ended employment in the past 24 months.

Thawing primary leukemia cells v1

This protocol is used to thaw primary cells. Key points are the addition of DNAse, which helps to preserve viability.

A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models

CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.

3D Genomics of Acute Meyloid Leukemia Reveals the Imbalance between DNA Methylation Canyon Interactions and Leukemic Specific Enhancer Network Interactions

Changes in 3D chromatin organization like enhancer hijacking are believed to the driver for disease development like leukemia. Here we performed high-resolution HiC assays on primary acute myeloid leukemia (AML) samples and cell lines to dissect the abnormal 3D chromatin organization in AML. Our data set covers 5 AML samples and 3 AML cell lines. This dataset includes the common genetic abnormalities in AML: MLL-rearrangement, NPM1 mutation, RUNX1 mutation, and IDH1/TET2 mutations. We have recently generated high-resolution map for normal human hematopoietic stem cells (HSC) (Zhang et al. Mole Cell. 2020). In comparison with the HSC 3D chromatin organization, we found TADs and loops are very stable in both primary leukemia samples and cell lines. Less than 5% of all TADs in HSC fuse in AML, mimicking the enhancer hijacking scenario. These fusion events do not cause the gene expression changes of genes in the fused TAD. Interestingly, in TET2 or IDH1 mutated AML blast, two-fold more TAD fusion events occurred in primary AML blast in comparison with RUNX1 and MLL-r leukemia, with a loss in the CTCF sites on the TAD fusion break point. We previously found in HSC, the Polycomb marked DNA methylation Canyons (DMC) form multi-Mb size long-range interactions. DMC interactions in general decrease in primary AMLs. AMLs with IDH1 or TET2 mutations shows the biggest reduction in DMC interactions. Hypermethylation in the DMCs is observed in the AML samples with IDH1/2 or TET2 mutations, suggesting DNA methylation level in DMCs controls DMC 3D interactions directly. In leukemia cell lines, the DMC interactions almost disappear, with further hypermethylation in DMCs. Compared with normal HSC, we found in AML, the AML-specific H3K27ac marked regions form leukemia specific loops and transcription stripes in both cell lines and primary samples. Particularly in MLL-r primary leukemias, we found broad H3K27ac covered, hyperacetylated domains (10kb to 200kb). 22 such hyperacetylated domains were identified and associated with leukemogenic genes such as SATB1, ZEB2 and HOXA. All these domains formed distinct 3D micro TAD in the MLL-r primary leukemia in comparison with the HSPC, and CTCFs are not located at the border of these domains. Taken together, suggest active leukemia specific transcription created new 3D genomic interactions which is independent of cohesion-CTCF mediated loop extrusion. Interestingly, in HOXA cluster, we found a geneless DMC 1.3MB upstream of HOXA switched from Polycomb binding site to active enhancer site in the leukemia cells. By applying CRISPR/Cas9 editing, we found this canyon is essential for survival of HOXA high expressing leukemia cell lines like OCI-AML3 and MV4:11. In summary, we found the 3D chromatin organization in human leukemia significantly alters in two opposite way 1. The significant loss of Polycomb marked DMC interactions caused by the DNA hypermethylation and 2. The leukemic specific hyperacetylated domains form its own distinct micro TAD and stripes in the 3D chromatin organization. Disclosures No relevant conflicts of interest to declare.

Group I p21-activated kinases in leukemia cell adhesion to fibronectin

P21-activated kinases (PAK) regulate many processes associated with cytoskeleton dynamics, including cell adhesion, migration, and apoptosis. PAK function is frequently altered in cancer, and PAK were proposed as therapy targets both in solid tumors and in hematological malignancies. However, current knowledge about PAK function in cell adhesion is mainly based on adherent cell models. Moreover, existing functional differences among the individual PAK family members are unsufficiently characterized.We measured expression of PAK group I members in leukemia cell lines and in primary leukemia cells, both on protein and mRNA levels. In functional assays, we analyzed the effect of two PAK inhibitors with different mechanisms of action, IPA-3 and FRAX597. Changes in cell interaction with fibronectin were monitored through impedance measurement and by interference reflection microscopy. Cytotoxic effects of inhibitors were assessed by Annexin V/propidium iodide test. PAK intracellular localization was analyzed by confocal microscopy.PAK2 transcript was dominant in cell lines, whereas primary leukemia cells also expressed comparable amount of PAK1, which was detected as two transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 transcript levels correlated with surface density of integrins β1 and αVβ3. PAK1-full, but not PAK2, was present in membrane protrusions. The inhibitors had partly opposed effects: IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death. Although many cells accumulated high FRAX597 amounts, low doses were sufficient to kill sensitive cells. FRAX597-induced cell death was fast in the MV4-11 cell line and in primary AML cells.Although PAK group I seem to be essential for leukemia cell adhesion and survival, and might thus serve as therapy targets, many PAK functions still remain to be attributed to individual isoforms and to their functional domains.

Dwjm-Adherence Induces Chemotherapy Resistance in Primary Acute Myeloid Leukemia By Altering Leukemia Cell Metabolism

Introduction: In previous work, we have demonstrated that culturing leukemia cell lines in decellularized Wharton's jelly (WJ) matrix (DWJM), the gelatinous material in umbilical cord tissues, resulted in chemotherapy resistance. To reduce the variability in the biochemical composition between different parts of the WJ matrix and to optimize our 3-dimensional (3D) DWJM-based extracellular matrix (ECM) model for in vitro culture of primary AML cells, we fabricated DWJM-derived porous disks with uniform architecture and pore-size by homogenization followed by lyophilization of human DWJM. Herein, we examine whether DWJM disks support primary leukemia cells and result in chemotherapy resistance. Methods: AML patient samples collected by leukapheresis were cultured in DWJM disks for one week. Non-adherent cells were first aspirated and adherent cells were separately isolated and assessed for viability, apoptosis, and colony forming unit (CFU). RNA sequencing was performed at the end of culture. Response to chemotherapy following treatment with doxorubicin was also assessed. For all studies, DWJM-adherent and non-adherent cells were compared to suspension culture controls. Metabolic pathway analyses were conducted using enzyme-linked immunosorbent assay (ELISA). One-tailed t-test was used for comparison between the groups. Results: Consistent with our prior studies, adherent DWJM cells demonstrated less apoptosis (P=0.027) and greater CFU activity with larger and more dense colonies (1 vs 4/50,000 cells, P=0.0008). Co-culture of primary AML samples with DWJM reduced doxorubicin induced cell death (P=.047) preserving CFU activity (3.3 vs 0.3/300,000 cells, P=.047) compared to treatment of suspension culture cells. To understand the mechanisms by which co-culture with DWJM enhanced leukemic progenitor function and therapy resistance, we performed RNA-Seq analyses. RNA-Seq data analysis from day 7 demonstrated significant upregulation of FAM83A and MIR34A and downregulation of BPI, ZNF521, NHLH2, CD69, FKBP14, PBX1, TANC1, GRIN2b, MYO6, INHBA, SA1008, CXCL1, A1009, BLNK, MMP9, BHLHE41, and CD9 in the adherent population compared to the suspension population (adjusted P-value <0.05). Ingenuity Pathway Analysis (IPA) identified glutamate receptor signaling as the top impacted canonical pathway, suggesting differences in the predominant metabolic process between the two culture conditions. Additionally, IPA showed that FAM83A was the most upregulated molecule, while MT-TH and MT-TW were two of the most down-regulated molecules in the adherent cells. The DWJM culture condition was associated with a significant increase in lactate (P=0.020) and significant reduction in glucose (P=0.005) in culture supernatants compared to suspension controls. Conclusion: We demonstrate that DWJM disks support primary leukemia cell survival. DWJM-adherent cells demonstrate chemotherapy resistance in association with induction of glycolysis. Based on our RNA Seq data we hypothesize that leukemia cell adherence to DWJM upregulates FAM83A, which functions in the epidermal growth factor receptor (EGFR) signaling pathway. FAM83A is known to control PI3K-AKT-TOR signaling cascade. M-TOR, in turn is known to activate HIF-1α pathway, which regulates glycolysis (Figure-1). Additionally, down-regulation of mitochondria-related molecules (MT-TH and MT-TW) is consistent with a switch from oxidative phosphorylation to glycolysis in the adherent cells. Further work is ongoing to further understand the role of glycolysis in primary leukemia cell chemotherapy resistance. Disclosures Lipe: Celgene: Consultancy. Aljitawi:The University of Rochester Medical Center: Patents & Royalties: Pending patent related to decellularized Wharton's jelly matrix; Medpace: Consultancy.

Natural Killer Cell Derived Exosomes As a Novel Therapeutic for Acute Myeloid Leukemia

Introduction: Natural killer (NK) cells play a critical role in the innate immune response through their capacity to lyse malignant cells without prior antigen-specific priming. NK cells have been evaluated for safety and efficacy in acute myeloid leukemia (AML), both in the transplant and non-transplant settings. Exosomes, small, 30-150 nm-sized extracellular vesicles originating from the endocytic compartment of parent cells, have recently emerged as a universal intercellular communication system. Exosomes are released by virtually all cells and carry proteins, lipids, and nucleic acids from the parent to recipient cells at short and long distances. The exosome molecular cargo reflects the content of the parent cell and is delivered to recipient cells in a membrane-protected vesicle. We hypothesize that exosomes produced by human activated NK cells carry the machinery necessary for the killing of leukemic cells. Methods: Venous blood (20-50 mL) was obtained from healthy donors (n=10). NK cells were isolated using Ab-based immunomagnetic selection from the recovered peripheral blood mononuclear cells. NK cells were cultured in the presence of interleukin-2 and interleukin-15, and NK-cell supernatants were used for exosome isolation by size exclusion chromatography. Protein levels, numbers and size (qNano), and exosome morphology (transmission electron microscopy) were determined. Exosome cargos were studied by Western blots and/or flow cytometry for NK cell activating and inhibitory receptors, immune inhibitory molecules and for perforin and granzyme B. Cytotoxicity of the NK cell-derived exosomes for K562 targets, AML cell lines (Kasumi, MLL-1) and primary leukemia blasts was measured using flow cytometry-based assays. Results: Activated human NK cells produced large quantities of exosomes. PKH-26-labeled NK cell-derived exosomes were avidly taken up by leukemic blasts. NK cell derived exosomes carried activating NK cell receptor NKG2D, natural cytotoxicity receptors, perforin, granzyme B, transforming growth factor beta (TGF-β), killer-cell immunoglobulin-like receptors and PD-1. NK cell derived exosomes mediated anti-leukemia activity against K562 targets, AML cell lines and primary leukemia blasts. Lysis of leukemic blasts by NK cell-derived exosomes was exosome concentration dependent. Conclusion: We report that NK cell derived exosomes have anti leukemia activity in vitro. These data provide a foundation for the future development of new therapeutic strategies using NK cell-derived exosomes for the elimination of leukemia. Disclosures No relevant conflicts of interest to declare.

Dynamics of Genetic Landscapes and Clonal Evolution between Patients and PDX Models in Acute Myeloid Leukemia

Introduction: Recent genome-wide studies of leukemia have revealed its clonal architecture and subclonal heterogeneity. Patient-derived xenograft (PDX) models are assumed to capture the cellular and molecular characteristics of human cancer and widely utilized for studying drug response. However, it has not been elucidated whether PDXs faithfully represent the genetic features of primary leukemia. In this study, we monitored the dynamics of somatic mutations in primary leukemia cells and PDX cells to evaluate their genetic stability and clonal selections. Methods: A total of 90 fresh bone marrow or peripheral blood samples from patients with hematological malignancies were intravenously transplanted into NOD/Shi-scid, IL-2Rγnull (NOG) mice at 0.2-15x106 cells per mouse. Targeted sequencing of 54 genes frequently identified in myeloid malignancies was performed in 40 paired genomic DNA of successfully engrafted AML patient and their PDX samples and 20 AML patients with engraftment failure. Clonal diversities were analyzed by comparing variant allele frequencies (VAF) of somatic mutations in 16 sets of serial samples from AML patients at diagnosis and subsequent relapse, and their PDX models. All patients provided written informed consent. Results: Sixty-five of 90 (72%) primary cells (AML; 60, ALL; 5) engrafted in NOG mice at the median of day 112 (29-549) post transplantation. Patient samples with successful establishment of PDX models included significantly higher number of infused cell counts (7.2 vs. 4.9 x 106 cells, P=0.002), higher percentage of blasts (56.2% vs. 38.7%, P=0.04) and harbored activating kinase gene mutations more frequently than those with engraftment failure (83% vs. 40%, P=0.001; Figure 1). Especially, FLT3 (53% vs. 10%, P=0.001), NPM1 (28% vs. 5%, P=0.04), RUNX1 (25% vs. 0%, P=0.01), IDH1 (20% vs. 0%, P=0.03) and ASXL1 (18% vs. 0%, P=0.05) mutations were significantly accumulated in successfully engrafted patients. In terms of the sensitivity to chemotherapy, patients with successful engraftment in NOG mice showed poorer 2-year overall survival than those without engraftment (36.1% vs. 100%, P=0.04; Figure 2). In 40 pairs of AML patients and their PDXs, a total of 172 genetic alterations (101 SNV, 39 frameshift, 32 inframe insertion/deletion) were identified. Mean VAF changes in PDXs compared with patients showed dominant elevation in driver gene mutations including FLT3 (+16.9%), CEBPA (+19.1%) and WT1 (+26.4%) demonstrating the expansion of clones carrying these mutations in PDXs, whereas those of ASXL1 (-2.2%), NPM1 (+4.8%) and DNMT3A (+6.0%) in PDXs were almost the same as primary patient samples, implicating that these mutations are founder events in clonal evolution. VAF of 26 pairs of patients' samples and their PDXs at relapse were more concordant than those of 12 pairs at diagnosis (r2=0.689 vs. 0.550). In 16 sets of AML patients' samples at diagnosis and relapse, and their PDX samples, clonal diversities between primary leukemia at diagnosis and their PDXs were classified into 2 subtypes. In 7/16 models (44%), minor clones detected in patients at diagnosis dominantly expanded to be major clones in their PDX models, whereas major clones persisted in similar VAF between primary leukemia and their PDXs in 9/16 models (56%). These subtypes of clonal diversity between primary leukemia at diagnosis and their PDXs were concordant with clonal evolution between patients at diagnosis and relapse in 12/16 models (75%). Furthermore, in 4/16 models (25%), clones selectively expanded in PDXs were detected as major clones in their patients' first relapsed samples predictively. Conclusions: Our findings indicate the existence of diverse clonal evolution in AML PDX models. Primary AML samples harboring treatment-resistant clones have higher potential of engraftment and growth in NOG mice. Furthermore, PDX models recapitulate the clonal evolution from diagnosis through relapse in treatment-resistant patients. Further understanding of expanded clones in PDX models could reveal the pathogenesis of clonal selection in AML patients. Disclosures Kiyoi: Bristol-Myers Squibb: Honoraria; FUJIFILM Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Celgene Corporation: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.