B-ALL Cells Research Articles

Abstract C055: Distinct epigenomic regulation of gene expression in Hispanic/Latino children with high-risk B-cell acute lymphoblastic leukemia

Abstract B-Cell Acute Lymphoblastic Leukemia (B-ALL) is the most common childhood malignancy. Hispanic/Latino (H/L) children have a 40% higher death rate than non-Hispanic white (NHW) children after correcting for socioeconomic factors. Recently, we identified a 3.5-fold increased incidence of deletion of one IKZF1 allele (IKZF1-del) and over 15-fold increased incidence of deletion of one IKZF1 allele concomitant with IGH-CRLF2 translocation (IKZF1-del/IGH-CRLF2) in B-ALL of H/L children. This provided a biological rationale for the worse prognosis of B-ALL in H/L children. The very high incidence (29% in all H/L children and 60% in ≥10yr old H/L children) makes IKZF1-del the most frequent genetic alteration that confers adverse prognosis in B-ALL in H/L children. The IKZF1 gene encodes a DNA-binding protein, IKAROS, which regulates transcription of its target genes via chromatin remodeling. The purpose of the study is to analyze the global epigenomic regulation of gene expression in H/L and NHW children with B-ALL and identify biological determinants of altered gene expression that result in worse prognosis for H/L children with B-ALL. /. B-ALL primary cells from 60 H/L and NHW children were analyzed with whole genome sequencing and gene expression profiling using RNA-seq. A subset of B-ALL from each group was analyzed for epigenomic changes by Reduced Representation Bisulfite Sequencing (RRBS), ChIP-seq, CUT&RUN, and CUT&Tag. Results showed that gene expression pattern in B-ALL of H/L children is quite different from B-ALL of NHW children. GSEA and Ingenuity pathway analysis identified oncogenic drivers of B-ALL in H/L children. B-ALL of H/L children have elevated expression of genes that promotestemness, inflammation, and with antiapoptotic activity. RBBS analysis showed a distinct DNA methylation pattern in B-ALL in H/L vs NHW children at promoters of a large set of genes that are involved in cellular signaling, RNA processing, and cellular proliferation. Presence of IKZF1-del results in the alteration of global epigenomic regulation of gene expression with altered enhancer and super-enhancer landscape. Epigenomic alterations due to IKZF1-del resulted in profound redistribution of enhancers and super-enhancers in B-ALL of both H/L vs. NHW children. This was associated with a distinct alteration in gene expression and uncovered that IKAROS regulates different sets of genes in B-ALL in H/L vs. NHW children. These data identified a unique oncogenic signaling that are responsible for aggressive course of B-ALL in H/L children. In conclusion, the multiomics analysis of primary B-ALL cells revealed a distinct global epigenomic regulation of gene expression between B-ALL from H/L and NHW children. This was demonstrated by alterations in DNA methylation and enhancer landscape. Presence of IKZF1-del has a different effect on gene expression in B-ALL of H//L vs NHW children. Results of the study provide a biological rationale for the worse prognosis of B-ALL in H/L children and identified novel therapeutic targets for this disease. Citation Format: Laura Budurlean, Bing He, Yali Ding, Diwakar Bastihalli Tukaramrao, Chingakham Singh, Joseph Schramm, Daniel Bogush, James Broach, Sinisa Dovat. Distinct epigenomic regulation of gene expression in Hispanic/Latino children with high-risk B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C055.

Repurposing GSK3B Small Molecule Inhibitors for Refractory Lymphoid Malignancies

Background: Small molecule GSK3B inhibitors were developed for solid tumors and neurological conditions, including Alzheimer's and Parkinson disease. Five GSK3B inhibitors achieved favorable safety and PK/PD profiles in a total of 22 Phase 1 and 2 clinical trials but no clinical response for any of these indications. GSK3B is the kinase that phosphorylates β-catenin to initiate its degradation, hence GSK3B small molecule inhibition results in rapid nuclear accumulation of β-catenin protein. Significance: Activating mutations in the Wnt/β-catenin pathway are common oncogenic drivers throughout all main types of cancer (n=64,812) but not found in B- and T-lymphoid malignancies (n=2,137). Studying tissue microarrays by β-catenin immunohistochemistry, lung, colon, breast, and melanoma specimen (n=57) showed consistently high levels of nuclear β-catenin, whereas B- and T-cell lymphomas (n=84) lacked β-catenin signal. Despite comparable mRNA levels, proteomic analyses revealed 60-400-fold lower β-catenin levels in lymphoid compared to epithelial tumors (n=1,389) suggesting that lymphoid malignancies rely on powerful mechanisms for β-catenin degradation, e.g. by GSK3B. Results: To study the effects of β-catenin activation in human cells, we induced expression of stabilized form of β-catenin in 18 lymphoid (B- and T-ALL, MCL, DLBCL, PTCL) and four myeloid leukemia cell lines. In B- and T-lymphoid cells, β-catenin accumulation compromised clonal fitness, colony formation and induced cell death, but increased competitive fitness and colony formation in myeloid leukemia cells. CEBPα-mediated reprogramming of B-ALL cells into a myeloid phenotype reversed the deleterious effects highlighting that effects of β-catenin-accumulation strictly depend on lymphoid lineage-identity. RNA-seq studies upon inducible activation of β-catenin in lymphoid cells, revealed transcriptional repression of Myc and Myc target genes as principal gene expression change. Unlike activating β-catenin:TCF7 complexes in epithelial cells, our global interactome studies in B- and T-ALL and lymphoma cells identified repressive β-catenin complexes with lymphoid-specific Ikaros zinc finger (IKZF1/2/3) proteins and several components of the NuRD complex including Mta1/2, Gatad2a/b, Chd4, HDAC1/2. Interactome and ChIP-seq analyses revealed that β-catenin together with lymphoid-specific Ikaros factors recruited repressive NuRD complex members to deacetylate H3K27 and repress MYC superenhancer (SE) regions. Genetic deletion of both IKZF1 and IKZF3 or HDRT-based mutation of Ikaros binding motifs within the Myc-SE enabled resistance to β-catenin accumulation. To leverage this previously unrecognized vulnerability of lymphoid malignancies, we examined five clinically approved GSK3B small molecule inhibitors that achieved favorable safety profiles at micromolar plasma concentrations in clinical trials for neurological disorders and solid tumors. Strikingly, LY2090314 and CHIR99021 were effective at low nanomolar concentrations in B- and T-cell malignancies that expressed lymphoid Ikaros factors. At IC50 concentrations of <5 nM, GSK3B-inhibitors induced massive accumulation of β-catenin, repression of MYC and acute cell death. In B-ALL cells with IKZF1-deletion, expression of the remaining IKZF3 factor was sufficient to retain full sensitivity to GSK3B-inhibition. Deletion of β-catenin, however, abolished the effects of GSK3B inhibitors and relieved suppression of MYC, demonstrating accumulation of β-catenin represents as central mechanism of action. Preclinical in vivo treatment experiments of refractory B-ALL and mantle cell lymphoma PDX validated small molecule GSK3B-inhibition as strategy to overcome conventional mechanisms of drug-resistance. Conclusions: B- and T-lymphoid malignancies not only lacked expression of β-catenin but critically depend on GSK3B for effective β-catenin degradation. Our interactome studies in lymphoid tumors revealed that β-catenin formed repressive complexes with lymphoid-specific Ikaros and NuRD complex factors. Strikingly, GSK3B small molecule inhibitors engage this new pathway at low nanomolar concentrations and demonstrated favorable safety profiles in Phase 1 and Phase 2 trials. Safety and efficacy of GSK3B-inhibitors opens up an immediate path for repurposing these agents towards refractory B-cell malignancies.

Computational Discovery and Validation of NAD+ Biosynthesis As Unique Vulnerability in B-Lymphoid Malignancies

Background and Significance: Glucocorticoids have been a mainstay in virtually all therapy regimens for B-lymphoid malignancies since the 1950s. Glucocorticoids selectively kill B-cell leukemia and lymphoma cells but have very limited effects on myeloid and epithelial cells. Likewise, inhibitors of B-cell signaling (e.g. ibrutinib) and selective depletion of B-cells (e.g. rituximab, CD19 CAR-T cells) have achieved important progress and are well tolerated. Drug discovery tool for selective targeting of B-cell malignancies: For these reasons, we developed an interactive computational tool ( lymphoblasts.org) to identify novel B-cell selective vulnerabilities based on integrated genetic and pharmacological compound screens ( Figure 1). Compound screening data from CTD and GDSC databases were reprocessed by fitting 4-parameter log-logistic dose response curves. Differential gene-dependency was also tested using DEMETER2 and Chronos scores, from Project Achilles RNAi and CRISPR datasets, respectively. To prioritize cell-type specific targets, compounds were ranked by average differential rank percentile of both compound-sensitivity and target-gene dependency. NAD+ biosynthesis as unique B-lymphoid vulnerability: Integration of genetic RNAi and CRISPR-based sensitivity data identified NMNAT1 and NAMPT as top-ranking B-cell selective targets that regulate NAD+ salvage and biosynthesis in the nucleus ( Figure 1). NAMPT, was previously identified as “pre-B-cell colony enhancing factor” and functions as rate-limiting enzyme in the salvage biosynthesis pathway of NAD+, which is critical for cellular energy metabolism. These genetic results were further corroborated by the identification of four small molecule inhibitors of NAMPT, with a striking B-cell-selective sensitivity profile. Compared to a panel of 1,078 myeloid leukemia and solid tumor cell lines, B-cell leukemia and lymphoma cell lines (n=98) were dramatically more sensitive to NAMPT inhibitors, including STF31, CAY10618, GMX1778 and Daporinad ( Figure 1). To verify these results based on the DepMap dataset and our drug discovery tool (lymphoblasts.org), we studied the effects of seven NAMPT inhibitors (CAY10618, FK866, GMX1778, OT82, STF118804, STF31 and KPT9274) on a panel of myeloid leukemia and solid tumor cell lines as well as PDX from patients with B-ALL and mantle cell lymphoma. Interestingly, three of these NAMPT inhibitors have demonstrated favorable PK/PD and safety profiles (NCT04281420, NCT04914845, NCT03921879). Genetic modeling of NAMPT-deficiency in B-cell malignancies. To model consequences of Nampt deletion in B-ALL, we transformed pre-B cells from Namptfl/fl mice with BCR-ABL1 and NRAS G12D oncogenes. Deletion of Nampt subverted competitive fitness of B-ALL cells, loss of colony-forming ability and cell cycle arrest. Additionally, we developed a conditional mouse model ( Namptfl/fl x Mb1-Cre) for B-cell-specific Nampt deletion, which resulted near-complete ablation of B-lymphopoiesis. Moreover, in vitro inducible deletion of Nampt caused rapid depletion and reduced colony formation in cultured bone marrow pre-B cells. Deletion of Nampt profoundly affected B-cell energy metabolism, significantly reduced FCCP-stimulated OCR and resulted in cell death owing to energy stress. Metabolomic studies of genetic Nampt-deletion using mass spectrometry revealed depletion of metabolites involved in amino acid metabolism, purine/pyrimidine metabolism, and the TCA cycle. Notably, phosphoserine was among the top-ranked downregulated metabolites, suggesting the involvement of one carbon pathway disruption in mediating energy crisis upon Nampt-deletion in B-ALL cells. Conclusions: These findings highlight that the classical ‘glucocorticoid paradigm’ can be extended to discover novel vulnerabilities, including NAD+ biosynthesis. Computational integration of genetic and pharmacological compound screens was particularly powerful in identifying previously unrecognized opportunities to leverage selective vulnerabilities of B-lymphoid leukemia and lymphoma. Our genetic studies corroborate the unique role of NAMPT and NMNAT1 in B-cell development and metabolic regulation. Further studies will focus on efforts to repurpose three clinically approved NAMPT small molecule inhibitors for the treatment of refractory B-cell malignancies.

Regulation of Alternative Splicing in B-Cell ALL By DYRK1A

DYRK1A, located in the Down syndrome critical region of chromosome 21, is a serine and threonine kinase that controls multiple cellular processes including apoptosis, cell cycle, transcription and signal transduction. We previously demonstrated that DYRK1A is required for maturation of B cells and is a therapeutic target in B-cell ALL, including both DS and non-DS subtypes. Key substrates within the B-cell lineage include D-type cyclins, FOXO1, and STAT3. Given that several studies have reported that DYRK1A phosphorylates various splicing factors and participates in splicing of Tau in neuronal cells, we investigated the contribution of DYRK1A to splicing in malignant B cells. We first treated B-ALL cell lines with two DYRK1A inhibitors, EHT 1610 and GNF 2133 and performed RNA-sequencing to detect changes in alternative splicing (AS). GO analysis of AS events showed RNA binding proteins among top differentially spliced genes. Of note, mis-splicing of RBM39 was one of the top differentially AS events associated with DYRK1A inhibition; this event involves inclusion of a “poison” exon that contains a premature termination codon and leads to a transcript predicted to be degraded by nonsense-mediated mRNA decay (NMD). We confirmed NMD by knocking down an essential NMD factor, UPF1, which stabilized the NMD isoform. The NMD isoform was also detected in B-ALL patient samples treated with DYRK1A inhibitors and in NALM6 cells in which DYRK1A was inducibly degraded. RBM39 has been reported to be a therapeutic target in AML, where cells with splicing factor mutations are more sensitive to loss of RBM39 than cells lacking splicing factor mutations. To determine whether RBM39 is a therapeutic target in B-ALL, we silenced RMB39 with shRNAs in vitro and in vivo. Genetic knockdown of RBM39 impeded B-ALL cell growth both in vitro and in xenograft models. Furthermore, inducible silencing of RBM39 hampered B-ALL progression in vivo evidenced by a significant increase in survival of mice engrafted with human B-ALL cells. We also found that indisulam and its analogs, which lead to DCAF15-mediated degradation of RBM39, suppressed the growth of B-ALL cells in vitro and in vivo. Importantly, the anti-tumor effect of RBM39 disruption extended beyond DS-ALL to Ph-like B-ALL, which continues to be an area of unmet medical need. We next analyzed the contributions of splicing factors that are known DYRK1A substrates to AS of RBM39. Knockdown of SRSF1 and SRSF2 led to increased inclusion of the poison exon even in the absence of DYRK1A inhibition, indicating that they normally function to exclude the exon. By contrast, knockdown of SF3B1 led to a reduction of the NMD isoform in the presence of EHT1610. Surprisingly, phosphorylation of SF3B1 by DYRK1A was not essential for the change in RBM39 splicing, as cells engineered to harbor two SF3B1 T434A phospho-deficient mutant alleles, did not show aberrant splicing. These results indicate that SF3B1, SRSF1, and SRSF2 are not the key substrates that mediate the effect of DYRK1A on alternative splicing. Next, given that DYRK1A is known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (Pol II) on Ser2 and Ser5, we investigated the contribution of Pol II phosphorylation to RBM39 splicing. IP-MS revealed an interaction between RNA Pol II and SF3B1, consistent with previous reports of co-transcriptional splicing. We found that inhibition of DYRK1A disrupted the colocalization of SF3B1 and RNA Pol II as well as with RBM39 RNA. However, DYRK1A inhibition had a transient effect on Ser2 and Ser5 phosphorylation, suggesting that cells have alternative pathways for Ser2/5 phosphorylation. Combining DYRK1A inhibition with inhibition or loss of CDK9, which also phosphorylates the CTD of RNA Pol II, led to a persistent decrease in RNA Pol II phosphorylation, marked increased NMD isoform and a profound reduction in RBM39 protein. Finally, we found that combining the CDK9 inhibitor dinaciclib with EHT 1610 synergistically inhibited growth of B-ALL patient cells in vitro and in vivo shown by significantly improved survival. Ectopic expression of RBM39 partially rescued the growth inhibition upon dinaciclib and EHT 1610 treatment suggesting downregulation of RBM39 is the key event in drug targeting. Together, our results reveal that DYRK1A controls RNA splicing by regulating the SF3B1-RNA Pol II interaction and that RBM39 is a therapeutic target in B-ALL.

Activin-a Increases B-Cell Acute Lymphoblastic Leukemia (B-ALL) Vesiculation and Modifies the Micro-RNA Cargo: Special Focus on MiR-491-5p

We recently demonstrated that ActivinA (ActA) is abundant in the leukemic bone marrow microenvironment where it exerts a pro-leukemic action by increasing the migratory and invasive properties of B-ALL cells. Extracellular vesicles (EV) are a new way of intercellular communication. They can be categorized in small-EV (sEV) and large-EV (lEV) based on size and origin. Similarly to migration, EV release is dependent upon cytoskeleton activation and membrane remodeling. EV play a prominent role in carcinogenesis, depending on the molecules they enclose, including miRNA that can downmodulate the expression of target mRNAs in recipient cells. The aim of the project was to study the impact of ActA on B-ALL vesiculation: quantity and miRNA cargo. Moreover, we investigated whether deregulated miRNAs could contribute to ActA-mediated leukemic progression. To investigate the role of ActA on B-ALL vesiculation, we used 697 and Nalm-6 cells lines. By means of nanoparticle tracking analysis, we demonstrated that after 24h, ActA significantly increased of 2.5 and 1.4 folds the number of sEV (p<0.0001) and lEV (p<0.0026, n=9) produced by 697 cells, compared to not stimulated cells (NS). A significant increase of both EV was also observed after 48h of ActA stimulation. The ability of ActA to promote B-ALL cell vesiculation was also confirmed on Nalm-6. Furthermore, we investigated the miRNA cargo of EV derived from 697 cells stimulated or not with ActA by OpenArray. After 24h, ActA significantly upregulated miR491-5p, let-7i-3p, and downregulated miR-639, miR135b-3p and miR139-5p. On the other hand, after 48h, ActA significantly upregulated miR15b-3p, let-7-e-5p, miR23b-3p and downregulated miR1236-3p, miR-18a-3p. We focused on miR491-5p, the most upregulated in ActA EV (3 folds at 24h), compared to NS (p<0.0001). ActA-mediated increase of miR491-5p was observed also in 697 cells (FC=1.5, p<0.0001, n=14). To investigate miR491-5p modulation also in Nalm-6 cells, they were stimulated or not for 24h and miRNA levels were evaluated in cells and their EV, isolated by ultracentrifugation. We preliminary observed that ActA increased miRNA expression of 479 folds in Nalm-6 EV (p=0.09, n=3), but only slightly in cells. Interestingly, absolute miRNA levels were higher of 4 folds in NS EV compared to NS cells (p=0.05), and showed an increasing trend in stimulated condition (FC=1552, p=0.09, n=3), suggesting that upon ActA stimulation, miR491-5p is upregulated and mostly encapsulated in EV. It has been described that miR491-5p promotes cell survival and proliferation in solid tumors. Indeed, we investigated whether ActA could impact on B-ALL cell viability through miR491-5p modulation. Indeed, 697 cells were stimulated every 3 days with ActA. Cell counts showed no differences between cells stimulated or not in the exponential growth phase. Interestingly, at plateau (day 7), ActA-stimulated cells were more viable (76%, range: 69%-80%) compared to NS cells (56%, range: 50%-66%, p=0.05, n=3). Alongside, we preliminary observed higher miR491-5p in ActA-stimulated cells (FC=3, n=1). It has also been reported that miR491-5p regulates chemoresistance in several cancers. Interestingly, by using Annexin-V/7-AAD assay, we demonstrated that ActA-stimulated 697 cells had a viability advantage of 11.3% (p=0.006, n=3), compared to NS cells, after 72h of ASNase-based chemotherapy, suggesting a chemoprotective role of ActA in B-ALL. Moreover, we observed that the combination of ActA and ASNase treatments induced the overexpression of miR491-5p compared to NS (FC=2.5, p=0.001) and to cells stimulated with ActA alone (FC=2.3, p=0.04) or ASNase alone (FC=1.4, p=0.004, n=8). To investigate the possible role of miR491-5p, we downregulated it into 697 cells by transfecting a miR491-5p inhibitor. Interestingly, under ASNase treatment, ActA still induced a viability advantage in cell transfected with a random inhibitor (negative control, 9.8%, p=0.006), comparable to untransfected cells. On the contrary, in miR491-5p inhibitor transfected cells, the viability advantage mediated by ActA decreased to 5.9% (p=0.03). Therefore, miR491-5p inhibition reduced of 30% the chemoprotective ability of ActA (p=0.04, n=3). In conclusion, these results suggest that ActA not only increases EV production, but also modifies their miRNA cargo and that miR491-5p could be one of the key players involved in ActA-mediated pro-leukemic action.

Suppressing IKZF1 Deletion-Mediated Activation of Ras Oncogenic Signaling By the Combination of Azacitidine and Flumatinib in High-Risk B-ALL

Background IKZF1 deletion is frequently observed in Ph + acute lymphoblastic leukemia (Ph + ALL), which results in poor prognosis and affects clinical efficacy, and possible resistance to tyrosine kinase inhibitors (TKIs). Ikaros transcriptionally regulates the gene expression of multiple oncogenic pathways including Ras/MAPK signaling. However, it is undefined about the role of IKZF1 genetic defects in the activation of Ras/MAPK signaling and their relations with the efficacy of therapeutic drugs in ALL. This study is to investigate the sensitivity of IKZF1-deletion-mediated activation of RAS signaling to the new combined therapy of TKI Flumatinib (FLU) and the Azacitidine (AZA) in B-ALL cells. Methods The patients' samples were obtained from Zhongda Hospital Southeast University with the approval of the institute's Ethics Committee. The gene expression was tested by qPCR or Western blot. CCK8 Cell proliferation, PI staining cell cycle analysis, and apoptosis assay (Annexin V-FITC/ PI staining) were performed in SUP-B15, the Ph + ALL cells with IKZF1 deletion treated with DMSO control, FLU only, AZA only, and FLU+AZA. The overlapped differential expression genes (DEGs）with the pathways (GO term, KEGG) analysis and gene interaction networks were achieved in the potential oncogenic genes in ALL through databases (OMIM, TTD, and GeneCards), and drug targets over that of SwissTargetPrediction, SuperPred, and SEA. Results AZA or FLU treatment induced the cell proliferation arrest of SUP-B15 cells in a dose-/time-dependent manner (Fig. 1A). The combination of IC50 AZA with various doses of FLU significantly enhanced the effect on cell proliferation arrest versus single-drug controls, and CalcuSyn analysis demonstrated their synergistic effect in the cells (Fig. 1B). Also, the combination significantly increased the G0/G1 phase arrest (Fig. 1C, D), and % of apoptotic cells (Fig. 1E, F) versus the single-drug controls. Consistently, the combination up-regulated the mRNA and protein levels of P53, BAX, and P21, but decreased the MDM2, BCL2, and CyclinE (Fig. 1G, H). A total of 118 overlapped DEGs were identified between the potential oncogenes in ALL and FLU-/AZA-associated genes, and they predominantly enriched in Ras/MAPK oncogenic pathways, in which PTPN11 is at the top of the pathways. Indeed, the combination significantly decreased the mRNA and protein levels of PTPN11 (Fig. 2A, B). PTPN11 is significantly higher expressed in 20 newly-diagnosed Ph +ALL patients versus 20 healthy bone marrow control (Fig. 2C), and it was validated in the GSE13159 dataset (Fig. 2D). PTPN11 high expression is significantly associated with lower overall survival and relapse-free survival compared to that of low expression in our cohorts (Fig. 2E). These data together indicate the combination exerts anti-tumor effect by suppressing RAS/MAPK signaling through down-regulation of PTPN11, and also PTPN11 promotes Ras activation in Ph + B-ALL. Moreover, about 80% of Ph +B-ALL patients have the IKZF1 deletion, our ChIP-seq data showed that IKZF1-encoded Ikaros protein binds to the promoter region of PTPN11 and CK2 inhibitor CX4945 as Ikaros function activator dramatically increase the Ikaros binding to the promoter of PTPN11 in B-ALL cells and Ikaros directly suppresses its promoter activity (Fig. 2F, G). We have reported that CX4945 demonstrated therapeutic efficacy for high-risk B-ALL by restoring Ikaros' function. Here, we observed that pre-treatment of SUP-B15 cells significantly increases the effect of AZA+FLU combination on cell proliferation arrest, and cell cycle arrest (Fig. 2H, I). Taken together, these results reveal a model that IKZF1 deletion-mediated Ras/MAPK activation via PTPN11 has the oncogenic role in Ph + B-ALL and the combination of AZA+FLU exerts the anti-tumor effect by targeting Ikaros/PTPN11/Ras oncogenic signaling. The model is summarized in (Fig. 2J). Conclusions The combination of AZA and FLU has a synergistic anti-leukemia effect on cell proliferation arrest and apoptosis in Ph +ALL cells with IKZF1-deletion by targeting Ikaros/PTPN11/Ras oncogenic signaling. Our data provide experimental evidence for a new potential combination of AZA with FLU in the therapy of Ph +ALL and highlight the likelihood of the novel combination in ALL patients.

High Dimensional Clustering and Identification of Immune Cell Hybrid Populations in B-ALL across the Disease Continuum

Introduction: Nearly half of adults with acute lymphoblastic leukemia (ALL) will experience relapse of their disease, and those with relapsed ALL carry a worse prognosis than those who remain in remission. The influence of the tumor immune microenvironment (TME) on relapse and survival outcomes in ALL is not well understood. Mass cytometry (CyTOF) enables immune profiling based on single cell protein expression and can identify phenotypic diversity of cells in the TME that cannot reliably be detected by standard flow cytometry. Recently, T-cell/monocyte hybrid cells have been discovered in patients with uveal melanoma and latent tuberculosis infections. However, cells expressing CD19, a surface antigen expressed on B-ALL cells, and markers of other cells in the TME including T cells and tumor-associated macrophages (TAMs), have not been described in ALL or other hematologic malignancies. The presence of CD3/CD163 hybrid cells in the TME may have predictive and prognostic implications for adults with ALL, as TAMs play a role in suppressing antitumor immunity which can lead to T-cell exhaustion. In this study, we identify the presence of multilineage hybrid cells in peripheral blood (PB) and bone marrow (BM) samples of patients with B-ALL alongside accompanying evolution of the TME at each disease stage. Methods: PB (N=16) and BM aspirate (N=16) isolates were collected from patients with B-ALL at diagnosis, relapse, or remission, as well as from healthy donors (HDs, N=7) for comparison of cellular profiles. A 28-marker CYTOF panel was designed to evaluate cell surface and intracellular markers of interest in the myeloid compartment. Antibodies were purchased pre-conjugated or conjugated in house based on manufacturer guidelines (Standard BioTools). Samples were barcoded with palladium isotopes prior to staining to reduce the impact of batch effect, and were then analyzed on a first generation CyTOF system (Standard BioTools). Following background attenuation and singlet gating in FlowJo (BD), the CATALYST R package enabled deconvolution and visualization of single cell data, including dimensional reduction techniques (tSNE) and clustering of all cell populations. An iterative approach was taken to determine a stable configuration of clusters across sample populations to protect from over-fragmentation of data, further informed by K-means clustering using the elbow method. Heatmaps were then generated based on median scaled expression of markers contained in each cluster. Results: The CyTOF panel identified several different myeloid cell populations in newly diagnosed, relapsed, and remission samples in both PB and BM compared to HDs. Most interestingly, quantifiable levels of a cluster of cells co-expressing CD19, CD3 and CD163 as well as a second cluster of cells expressing CD19 and CD163 were detected in newly diagnosed and relapsed patients, but not in HDs. A single patient with the largest proportion of these cell populations in their PB at the time of relapse presented with an aggressive leukemia variant and relapsed multiple times prior to succumbing to illness. On investigation of BM aspirates, the triple positive hybrid population was found in a similar distribution amongst the newly diagnosed and relapsed patients, as well as affecting one patient who achieved remission. This patient also had a highly aggressive leukemia that was refractory to chemotherapy and immunotherapy, and only achieved this remission following CAR-T cell therapy. She subsequently underwent stem cell transplant and is in remission. Conclusions: Our findings add to the literature highlighting the utility of high parameter single cell capture techniques in detecting rare cell populations in the leukemic TME. Further, we identified two unique hybrid cell populations that are reliably detected across different disease stages in patients with B-ALL. Importantly, these populations persisted following stringent gating protocols to rule out doublets and debris. Although our sample size was too small to demonstrate a prognostic impact of these hybrid cell populations in patients with B-ALL, the identification of these populations alone is a significant finding. Further investigation and patient sample accrual are warranted to better understand the role of these cell types in tumor biology, which may pave the way towards new prognostic models that can leverage well-established immune monitoring techniques.

Mitochondrial Functional Profiling Links Apoptotic Sensitivity to Treatment Outcomes Based on Age and Cell Lineage in Acute Leukemia

Background Survival outcomes in paediatric T and B cell acute lymphoblastic leukemia (ALL) are far superior to acute myeloid leukemia (AML). In addition, children with ALL and AML have better response rates than adults. While the basis for these differences in therapy outcomes is multifactorial, it is poorly understood. The majority of anticancer therapies activate apoptosis signalling to target cancer cells. The balance between pro-survival (BCL-2, BCL-XL and MCL-1) and pro-apoptotic proteins as well as evasion of apoptosis, plays a key role in cancers. Dependencies to these anti-apoptotic pro-survival proteins is a hallmark of haematopoietic malignancies and can be exploited with novel agents especially by using Venetoclax (BCL-2 inhibitor) and Navitoclax (BCL-2 and BCL-XL inhibitor) that are now available in the clinic. We hypothesized that difference in apoptosis threshold (priming) based on cell lineage and patient age offers key functional insights into the differences in outcomes as well as gives us an in vitro window to assess inherent BCL-2 or BCL-XL dependencies that can be targeted. Methods We prospectively and retrospectively examined functional regulation of apoptosis by performing a BH3 profiling assay on leukemia patient specimens from two tertiary cancer centres in Singapore. Since BCL-2 family consists of different anti-apoptotic proteins, we asked whether protection from specific pro-survival BCL-2 family proteins such as BCL-2, BCL-XL or MCL-1, predicted by sensitivity to BAD, HRK and MS-1 peptide, respectively, contribute to overall difference in apoptotic sensitivity between lymphoid and myeloid precursors. We also aimed to determine dominant factors that govern differences in clinical chemosensitivity to lymphoid compared to myeloid tumours. Blasts isolated via flow cytometry from bone-marrow aspirate (BMA) or peripheral blood were exposed to a variety of pro-apoptotic BH3 peptides to measure mitochondrial outer membrane permeabilization (cytochrome c release). The percentage of cytochrome c loss with each BH3 peptide at different concentrations was normalized to the cytochrome c release from negative control DMSO wells. Assays were performed in triplicate. Alamethacin was used as positive control. Flow cytometric data was acquired with a BD analyser, analysed with FlowJo and plotted in GraphPad Prism. Results Initial analysis of seventy one patient samples are presented here (40 AML, 17 T-ALL, 12 B-ALL and 2 MPAL). Lymphoblasts showed significantly increased apoptotic sensitivity as measured by Bim response compared to myeloblasts (p<0.001). We found lymphoblasts to be significantly more dependent on BCL-2 and BCL-XL compared to myeloblasts (p<0.001). Higher MCL-1 dependence was also noted in B-ALL cells as compared to AML (p<0.05). We next asked if apoptotic sensitivity measured by mitochondrial priming could explain higher chemosensitivity of pediatric over adult patients. Indeed we noted increase in overall priming in paediatric (N=26) as compared to adults (N=36) irrespective of cell of origin (P<0.001). Within the AML cohort, we found pediatric AML (N=8) to be more sensitive to Bim peptide compared to adults (N=32), as well as showed closer proximity to apoptosis (p<0.05). Of note there were no differences in overall priming or selective dependencies between diagnosis and relapse/refractory (R/R) patients in AML or ALL cohorts. By using blasts of AML and +ALL patients (N=15), we found that increased mitochondrial priming response to HRK+MS1 peptides (which infer BCL-XL and MCL-1 dependence respectively) inversely correlated with achievement of remission with venetoclax based therapy (p<0.001). Mitochondrial sensitivity to MS-1 peptide, increased from pre-treatment to on treatment to relapse (N=3/3 patients) in sequential samples for three patients suggesting an opportunity for the use of MCL-1 inhibition in relapse setting. Conclusion Our study shows increased overall priming in lymphoblast as compared with myeloblast and in pediatric as compared to blasts from adult leukaemia specimens. These findings offer a functional window to further explain differences in outcomes in leukaemia in different ages as well as lineages. Innovative trial designs incorporating cellular vulnerabilities to anti-apoptotic proteins using functional BH3 profiling as an accessory biomarker will enable improved treatment outcomes with reduced toxicity.

Integrating Proteomics and Functional Genomics to Identify Targets for Improving Glucocorticoid-Based Treatment of B-Lymphoblastic Leukemia

Glucocorticoids are a critical component of successful combination chemotherapy regimens for B-lymphoblastic leukemia (B-ALL). Despite advances in immunotherapies, glucocorticoid-based chemotherapy continues to be the essential frontline treatment of patients with B-ALL. This approach has led to high cure rates but at the risk of both acute, potentially life-threatening complications and chronic toxicities. Additionally, glucocorticoids remain ineffective for some patients with B-ALL, which predicts poor outcomes. To achieve better outcomes and reduce the toxicity of treatment, we take a functional genomics approach to understand how glucocorticoids induce cell death and identify strategies to make them more effective. Glucocorticoids work through the glucocorticoid receptor (GR), a ligand-activated transcription factor, to induce cell death in B-ALL. By integrating gene expression and RNA interference screening, we have identified both effector genes (genes whose regulation by glucocorticoids contributes to B-ALL cell death) and sources of resistance that can be targeted to improve treatment (Kruth et al Blood 2017). This identified the network of BCL-2 family proteins (e.g., BCL2, BCL2L11, BMF), whose regulation contributes to apoptosis in B-ALL. Surprisingly, it also showed that suppression of B-cell development genes (e.g., EBF1, PAX5, IL7R) contributes to B-ALL cell death. However, only a fraction of the genes that affect the glucocorticoid sensitivity of B-ALL are effector genes. We therefore sought to identify functional interaction partners of GR, defined as proteins that physically interact with GR and influence glucocorticoid sensitivity. To identify proteins that physically interact with GR, we used proximity labeling. We fused an ascorbate peroxidase domain (APEX2) to the N-terminus of GR and expressed it REH cells, a B-ALL cell line that does not express GR protein. After verifying that APEX2-GR restored glucocorticoid sensitivity in the REH cells, we performed proximity labeling in the presence and absence of dexamethasone. After optimizing these reactions, cells were lysed and labeled proteins were isolated using magnetic streptavidin beads and identified by mass spectrometry. We were able to detect more than 4,000 proteins that interact with GR, including hundreds that are enriched upon addition of dexamethasone. To identify functional interaction, we intersected the proteins enriched upon addition of dexamethasone with proteins shown by us to modulate glucocorticoid sensitivity in B-ALL (Kruth et al Blood 2017). Supporting our working model, we found functional interaction partners that are involved in B-cell development and apoptosis. We found that GR interacts with EBF1 and MEF2C, B-cell development transcription factors that restrain glucocorticoid-induced cell death. This indicates that GR activity and glucocorticoid sensitivity is directly blunted by B-cell development, and further supports the model that glucocorticoids restrain B-cell development. The mechanism of how these interactions influence glucocorticoid sensitivity will guide rational targeting to improve treatment of B-ALL both in general and in different B-ALL subtypes. Additionally, the mitochondrial voltage dependent anion channel 2 (VDAC2) also interacts with GR in a dexamethasone-dependent manner and is integral to glucocorticoid-induced cell death. VDAC2 is important for nucleating and perhaps triggering pore formation through BAK and BAX. How GR influences this activity is not known but suggests a non-genomic role of GR in glucocorticoid-induced cell death. In summary, integration of functional genomics and proteomics provides rich data sets that will help understand the mechanism of glucocorticoid-induced B-ALL cell death. Targeting of non-canonical (B-cell development) and non-genomic (VDAC2) functions of GR will allow for optimization of glucocorticoid-based chemotherapy regimens.

Dynamic Recruitment of Inhibitory Complexes Controls Oncogenic Signaling in B-Cell Malignancies

Background and Significance: Initiation of B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. High expression levels on the surface of B-ALL cells are associated with poor clinical outcomes in pediatric (COG P9906) and adult (ECOG E2993) B-ALL cohorts. In DLBCL, aggressive disease is associated with CD25-inactivation by shedding and high serum levels of soluble CD25. Results: Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ, immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors and inhibitory phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ and phosphatases, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early stages of B-cell development (e.g. B-ALL) CD25-loss induced hyperactivation of oncogenic signaling followed by cell death. In contrast, genetic deletion of CD25 in models for mature B-cell lymphoma accelerated proliferation of malignant cells. Biochemical and interactome studies revealed a critical role of CD25 in feedback regulation of oncogenic signaling in B-cell malignancies: Signaling from BCR-ABL1 and RAS-oncogenes in B-ALL as well as oncogenic BCR-signaling in DLBCL induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268). Importantly, CD25-phosphorylation stabilized its expression at the cell membrane and opposed both inactivation by CD25-shedding and internalization. Moreover CD25-S 268 tail-phosphorylation enabled CD25 to recruit inhibitory receptors, including CD22, that bear ITIM-motifs for the activation of inhibitory phosphatases (e.g. SHP1, Figure 1). Thereby, the positively charged cytoplasmic tail of CD25 engages negatively charged residues in the cytoplasmic tails of the BCR signaling chain CD79B and the inhibitory CD22-ITIM. Molecular dynamics simulations revealed that the ternary complex between CD25, CD79B and CD22 enables recruitment and activation of the phosphatase SHP1 within reach of its substrate, the BCR signaling chain CD79B. Thereby, the SH2-domain of SHP1 engages the CD22-ITIM motif, while its phosphatase domain makes contact with the CD79B-ITAM to dephosphorylate and terminate BCR-signaling. This complex, while engaging and activating SHP1, is held together by CD25 ( Figure 1). To test functional consequences of destabilization of this complex, we generated a knockin mouse model carrying a single point mutation (S268A) in the cytoplasmic tail of CD25. The CD25 S268A knockin mutation abolished recruitment and activation of SHP1. In addition, the S268A-mutant failed to assemble CD22 and CD79B in complex with SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function and failure to terminate oncogenic signaling resulted in constitutive activation of ERK and NF-κB, autonomous Ca 2+-oscillations. Hyperactivation of oncogenic signaling resulted in exhaustion and cell death in B-ALL cells, as opposed to excessive proliferation and acceleration of disease in mature B-cell lymphoma. Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, these findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and provides an explanation for the common feature of CD25 shedding and high serum levels of functionally inactive soluble CD25 in aggressive B-cell lymphomas.

Single-Cell RNA Sequencing Facilitates Study of Cancer Mechanisms across Pediatric Leukemias

Leukemia is the most common pediatric cancer, diagnosed in over 3,000 children annually in the US. While the prognosis for many pediatric leukemias has significantly improved with the addition of DNA sequencing to the diagnosis and treatment selection process, the molecular mechanisms driving some cases remain inscrutable. In this study, we performed single-cell RNA sequencing on a cohort of pediatric leukemia samples (n = 72) to explore intra- and inter-patient heterogeneity across multiple types of malignancies: acute megakaryoblastic leukemia (AMKL, n = 1), acute myeloid leukemia (AML, n = 14), acute promyelocytic leukemia (APL, n = 1), B-cell acute lymphoblastic leukemia (B-ALL, n = 32), anaplastic large cell lymphoma (ALCL, n = 1), myelodysplastic syndrome (MDS, n = 1), mixed phenotype acute leukemia (MPAL, n = 2), Ph-like B-cell acute lymphoblastic leukemia (Ph-like, n = 8), and T-cell lymphoblastic leukemia (T-ALL, n = 12). Using the 10x Chromium system, we generated single-cell RNA sequencing data for 183,321 cells from cryopreserved blood and bone marrow samples collected from patients at diagnosis (n = 65), remission (n = 3), and/or relapse (n = 4). Sequencing data were aligned to GRCh38 and counted using cellranger-6.0.1, with downstream analysis performed using Seurat 4.0.2 in R 4.0.3. Consistent with prior single-cell studies of leukemia, we observed a high degree of inter-patient heterogeneity: cancer cells from each patient showed distinct transcriptional signatures (Figure A; circled clusters indicate mature cells of each type clustering together, while other clusters correspond to leukemic cells from a single patient). Regardless of leukemia type and cell of origin, many patient samples contained cells most transcriptionally similar to hematopoietic stem cells (HSCs; Figure B). At diagnosis, the proportion of HSCs-like cells ranged from 7% in patients with MPAL to 46% in AML. Samples collected at relapse showed a striking increase in the proportion of HSC-like cells in both B-ALL (97%, compared to 45% at diagnosis) and Ph-like (92%, compared to 16% at diagnosis). Comparative analysis of this cohort additionally revealed marker genes specific to each leukemia, reflecting the diverse cell of origin and molecular mechanisms driving each cancer, including PRDM2 and ID3 for B-ALL, PRDM16 and CSF3R for AML, as well as SKI and PTPRC for MPAL. In this study, we use single-cell RNA sequencing of a large pediatric leukemia cohort to gain novel insights into the transcriptional drivers and heterogeneity of leukemias. Future work will extend these observations to explore transcriptional signatures associated with prognosis, both common and unique to individual cancers. In the long term, the findings from this research will facilitate improved diagnosis, patient stratification, and treatment selection for pediatric patients diagnosed with leukemia.

CDK12/13: A Promising New Therapeutic Target in ALL

Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy in children and young adults. Upfront resistance to glucocorticoids used as first-line treatment in most regimens confers a poor prognosis for patients with ALL. Of patients who relapse, only half achieve long-term survival. Here we demonstrate that inhibition of cyclin dependant kinases 12 and 13 (CDK12 & CDK13), which are modulators of RNA polymerase II, has a robust synergistic anti-leukemia effect with dexamethasone, in both B-ALL and T-ALL cell lines as well as primary B-ALL patient-derived bone marrow samples. To understand the mechanism underlying the steroid resistance in ALL, we first reanalyzed shRNA screen performed by Poulard et al. This revealed that CDK13 silencing sensitized leukemic cells to dexamethasone. CDK12 is located on chromosome 17q12, containing oncogenic features and genetic alterations in various tumors. CDK12/13 inhibitor exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft mouse model. It was shown that the inhibition resensitizes HER2+ breast cancers to HER2-tyrosine kinase inhibitors via suppressing PI3K/AKT in patient-derived organoids in vitro and cell-derived xenograft or patient-derived xenograft in vivo. Forgoing research demonstrates that CDK12/13 inhibition silences transcription of DNA damage response (DDR) genes. In our study, however, we haven't observed a rise in γH2AX, a marker of DNA damage, after CDK12/13 inhibition in ALL cell lines. What we did note was a marked enhancement expression of BIM and SGK1, steroid responsive genes and pro-apoptotic factors, after CDK12/13 inhibition (Figure A). ALL cell lines exhibited high susceptibility to CDK12/13 inhibition with two chemically divergent inhibitors, SR-4835 and THZ-531. We also showcased their synergistic interactions with dexamethasone using flow cytometry and synergyfinder ZIP score analysis (Figure B). Moreover, in the GSEA comparison of ALL cell lines with and without synergy between CDK12/13 inhibitors and dexamethasone, our data underscored significant enrichment in cholesterol biosynthesis-related genesets in cell lines manifesting this synergistic effect. On the other hand, we observed resistance to SR-4835 in cell lines with high enrichment scores in NOTCH, a signaling pathway implicated in about 80% of relapsed T cell ALL/LBL cases. Additionally, new data on mice xenotransplanted with B-ALL CNS-derived patient cells, link upregulated cholesterol biosynthetic pathways to nervous system involvement in childhood ALL. Upon administering CDK12/13 inhibitors, we observed a cell cycle phase shift resulting in G1 arrest and increased apoptosis, whereas human peripheral blood mononuclear cells (PBMCs) from healthy individuals remained unaffected by this treatment. As we continue to decode the pathways through which CDK12/13 inhibitors render ALL cells more susceptible to dexamethasone, our next step includes silencing CDK12/13 using short hairpin RNA models in cell lines and consequently their RNA sequencing. This approach may mitigate the influence of secondary pathways, paving the way for enhanced treatment strategies for relapsed ALL by unmasking the underlying molecular events.

Targeting EYA1 Activity in MLL- Rearranged Leukemia

The Mixed Lineage Leukemia gene (also known as MLL or KMT2A) is involved in chromosomal translocations in a subtype of acute myeloid (AML) and lymphoblastic leukemias (ALL). MLL translocations have been identified with more than 80 partner genes, and the most common AML translocation partner is MLLT3 (AF9). MLL-rearranged (MLL-r) leukemia is associated with a sudden onset, aggressive progression, and poor prognosis in comparison to non-MLL-r (MLL-nr) leukemias, and there is a need for more effective therapies. Previous work determined that AF9 directly interacts with multiple different regulatory proteins, including the BCL6 corepressor (BCOR). An AF9 point mutation (E531R) was identified that selectively disrupts BCOR binding to AF9 and MLL-AF9. The loss of BCOR binding to MLL-AF9 abrogated its leukemogenic ability in a mouse transplant model. RNA-seq analysis determined that of the MLL-AF9 direct target genes, EYA1 was the most down-regulated in the MLL-AF9 (E531R) point mutant-transformed cells. Addback of EYA1 into MLL-AF9 (E531R) cells partially rescued its leukemogenic ability, suggesting that EYA1 has an important role in MLL-AF9 leukemogenesis. EYA1 is a transcriptional coactivator and protein tyrosine phosphatase that is expressed during embryonic development and its re-expression has been implicated in several cancers, including MLL-r leukemia. The objective of this study is to investigate the in vitroand in vivo effects of EYA1 inhibition in MLL-AF9 transformed murine bone marrow stem/progenitor cells and in human MLL-rearranged (MLL-r) and non-rearranged (MLL-nr) leukemia cell lines. Cells were treated with small molecule inhibitors at varied concentrations and time points. Effects of inhibition were assessed with viability, cell morphology, cell cycle, surface and intracellular marker expression analyses, and in vivo transplant studies. EYA1 inhibition reduced cell viability by 50% - 90% in MLL-AF9 transformed murine bone marrow (mBM) cells and multiple human AML cell lines. EYA1 inhibition did not affect some human B-ALL cell lines, which also express undetectable levels of EYA1, indicating a correlation between the response to EYA1 inhibition and EYA1 expression. In MLL-r and MLL-nr cells, cell cycle analysis indicated a reduction in cells entering S-phase, stalling in G0/G1, with EYA1 inhibition as well as an induction in cellular senescence. Inhibiting EYA1 activity induced cell morphology changes toward a more differentiated state in MLL-AF9 mBM and several MLL-r and -nr cell lines. To confirm these qualitative results, cells were stained for myeloid differentiation markers, and cell surface expression of CD11b, Gr1, and CD14 increased in a dose- and time-dependent manner in all cell types, while HSC markers CD117 (c-KIT) and CD34 were reduced in MLL-AF9 mBM and human AML cells, respectively. In assessing the mechanism of EYA1 in MLL-r leukemia, MLL-AF9 cells treated with EYA1 inhibitors showed increased RNA pol II CTD Tyr1 phosphorylation levels (known EYA1 substrate) compared to vehicle-treated cells. Thus, unregulated EYA1 phosphatase activity targeting RNA Pol II CTD may contribute to leukemia development. Mice that received MLL-AF9 mBM cells treated with an EYA1 inhibitor had a significantly decreased rate of disease progression and increased survival compared to control. Bone marrow cells isolated from these mice showed increased expression of myeloid differentiation markers CD11b and Gr1 compared to vehicle-treated mice, confirming that EYA1 activity contributes significantly to leukemogenesis. The inhibition of EYA1 induces leukemia cell differentiation, cell cycle arrest and senescence, and cell death. Ongoing experiments include additional in vivo murine leukemia, further mechanistic analyses, and combinatorial experiments to evaluate the efficacy of a multi-target approach with EYA1 inhibition and other targeted-therapies. This work may provide a promising, novel therapeutic approach to MLL-r and MLL-nr leukemias that will help improve patient outcomes.

Enhanced T Cell Function of PTPN2 Deleted CAR-T Cells Comes at a Cost: PTPN2 Knockout CAR-Ts Secrete More Cytokines and Demonstrate Increased Cytotoxicity, but Exhibit More Severe CRS and Icans in a Non-Human Primate Model

Background: Despite the success of CD19 CAR-T cells (CAR-Ts) in inducing remission, relapse remains a major issue. Previous studies have shown that upregulation of negative T cell regulators can contribute to CAR-T failure, and that deletion of these regulators can enhance CAR-T efficacy. A potent negative regulator is PTPN2, which inhibits T cell function by modulating T cell receptor and cytokine signaling pathways. While murine models have shown that PTPN2-knockout T cells improve tumor control, these models fail to reliably predict clinical efficacy or toxicity. To improve this predictability, we investigated PTPN2 KO CAR-Ts in primary human cells and in a NHP model of B-cell directed CAR-T therapy. Methods: CRISPR/Cas9-mediated PTPN2 deleted (‘KO‘) human CD19 CAR-Ts were assessed for phenotype/function using cytokine secretion and cytotoxicity assays. PTPN2 KO NHP CD20 CAR-Ts were administered to lymphodepleted NHPs using our established model and a dose escalation with Level 1: 6x10 4, Level 2: 6x10 5, Level 3: 3x10 6, and Level 4: 6x10 6 CAR-T/kg, followed by assessment for CAR-T expansion, B cell aplasia, CRS and ICANS. Results: PTPN2 KO was successfully achieved in human CAR-Ts (83.8 + 5.2% deletion). IFNγ production was significantly higher in PTPN2 KO CD19 CAR-Ts vs control cells (35.1 + 0.97% vs 23.4 + 0.98%, p=0.001). In cytotoxicity assays targeting the B-ALL cell line NALM6, PTPN2 KO CD19 CAR-Ts demonstrated significantly increased killing vs control cells (for example, at E:T ratio 1:1 59.6 + 9.8% vs 42.5 + 12.9% p = 0.03). We evaluated the proliferation, efficacy, and toxicity of PTPN2 KO CD20 CAR-Ts in 5 NHPs. The CAR-T PTPN2 KO rate ranged from 63-91%. During dose escalation, no expansion of CAR-Ts was observed at dose levels 1 and 2 (n=1 for each, Fig 1). However, all animals infused at dose level 3 (n=3, 3x10 6 CAR-Ts/kg) demonstrated CAR-T expansion (max expansion 18.6 + 6.8% CAR-Ts) and B cell aplasia, and all animals developed laboratory and clinical signs of CRS and ICANS ( Fig 1). One animal at dose level 3 experienced CRS on day +4 that was severe enough to require tocilizumab, with symptoms subsequently improving. On day +6, this animal also developed ICANS, including ataxia and muscle weakness, requiring 1 dose of Dexamethasone (1mg/kg), after which symptoms resolved. One animal received dose level 4 (6x10 6 CAR-Ts/kg). This recipient displayed earlier and higher expansion of CAR-Ts (day 3: 16.7%, day 7: 56.3% CAR-Ts, Fig.1), and developed severe CRS (with elevated CRP, LDH (not shown) and Ferritin, Fig.1) and ICANS that were unresponsive to treatment with 2 doses of tocilizumab and multiple doses of dexamethasone, with this recipient meeting humane euthanasia endpoints. Importantly, previous animals (n=5) receiving 6-12 x 10 6PTPN2 expressing (WT) CAR-Ts/kg, did not develop severe CRS nor ICANS (and did not require Toci or Dex). This suggests that PTPN2 KO CAR-Ts are associated with augmented toxicities. An extensive terminal analysis of the Dose Level 4 animal was performed; pathology demonstrated widespread cerebral edema and CAR-T CNS infiltration. Because the CAR-T infusion contained both PTPN2 KO and PTPN2 WT CAR-Ts, an assessment could be made for enrichment of PTPN2 KO CAR-Ts in the blood and brain parenchyma. Compared to the infused product (63% KO CAR-Ts, Fig.2), there was enrichment for KO CAR-Ts in the blood (72% KO CAR-Ts) and the brain parenchyma (84% KO CAR-Ts, Fig 2). This suggests increased expansion and CNS infiltration of PTPN2 KO versus PTPN2 WT CAR-Ts. Flow cytometry analysis of the PTPN2 KO CAR-Ts from the blood at maximum expansion revealed high expression of the proliferation and activation markers Ki67 and GZMB (84.6%, 74.5% of all CAR-Ts). Of note, at Dose Level 3, neither CAR-T persistence (15 + 2 days vs 20 + 5 days) nor duration of B cell aplasia (41+ 5 days vs 37 + 3 days) were increased with PTPN2 KO CAR-Ts vs historic controls infused with PTPN2 WT CAR-Ts. Conclusion: We demonstrate that PTPN2 KO CAR-T cells possess increased effector function, proliferation, and CNS infiltration versus PTPN2 WT CAR-Ts. However, this enhanced efficacy was associated with increased severity of CRS and ICANS, emphasizing that careful attention to the dose of PTPN2 KO CAR-Ts would be important. These results also highlight the utility of evaluating gene-modified CAR-Ts in clinically relevant animal models, to thoroughly assess their efficacy and toxicity profiles.

Disinhibition of T Cell Activation Via CD5 Knockout Is a Universal Strategy to Enhance Adoptive T Cell Immunotherapies

Introduction: Adoptive cell therapies (ACT) are currently revolutionizing cancer treatment. In particular, chimeric antigen receptor (CAR) T cells have demonstrated unprecedented responses against aggressive B cell malignancies with six FDA-approved products either targeting CD19 or BCMA. Other strategies for solid cancers such as T cell receptor (TCR)-redirected or tumor infiltrating T cells will soon become commercially available for melanoma and other cancers. However, despite this exciting new wave of ACT, the majority of patients will ultimately fail treatment, even in the most potent products such as CART19. One key mechanism of failure is the inability of adoptively transferred T cells to fully activate and function in the tumor bed. Thus, there is a dire need to develop effective strategies to enhance T cell activation to improve long-term responses in patients. Most of the current strategies are focused on reducing T cells' exhaustion, but another avenue is to study early T cell activation as a key step for long-term efficacy. We sought to define a broadly applicable strategy to disinhibit T cell activation by focusing on CD5, a transmembrane protein shown to negatively regulate T cell activation and proliferation through mediators such as SHP-1, CBL, and CBL-B ( Fig. 1A). We previously presented the preliminary preclinical results of CD5 knocked out (KO) anti-CD19 CAR T cells (CART19) ( Blood (2020) 136 (Supplement 1): 51-52.) and demonstrated how CD5 KO dramatically increased T cell proliferation and enhanced tumor control. Here, we translate this effect to enhance ACT in multiple models including CAR T and TCR-redirected cells. We also compared early disinhibition of T cell activation (CD5 KO) compares with the highly studied PD1 KO to assess the translational potential of this strategy. Methods and Results: We initially tested CD5 KO CAR T cells in a model of pancreatic ductal cancer adenocarcinoma (PDAC; AsPC1 cell line). We deleted CD5 in anti-mesothelin CART cells (CARTmeso) using a clinically relevant CAR construct (M5 clone) (NCT03054298). CD5 KO CARTmeso cells demonstrated enhanced tumor control (by both tumor volume and bioluminescence) and prolonged overall survival ( Fig. 1B). As observed in the CART19 model, CD5 KO T cells showed increased expansion and persistence. Furthermore, CD5 KO CARTmeso cells established prolonged immune memory, as long-term surviving mice cleared rechallenged tumors (day 74). To study the effects of CD5 KO in TCR-redirected cells, we generated TCR α constant (TRAC) KO T cells further transduced with TCR-GP100 lentivirus and compared these to cells with a double KO of both TRAC and CD5 using the GP100+ melanoma cell line DM6. TRAC CD5 KO TCR-GP100 cells showed significantly reduced GP100+ DM6 tumor growth in vitro when compared to TRAC KO TCR-GP100 cells using a live imaging system, suggesting that CD5 KO could enhance the efficacy of adoptive T cell therapies as a whole. Immune checkpoint inhibitors targeting the PD1:PDL1 axis are routinely used in the clinic. More recently, deletion of PD1 in adoptively transferred T cells has shown feasibility in the clinic and there are currently over 30 clinical trials testing PD1 KO or inhibition in CART therapy for cancer. We, therefore, aimed to compare this established strategy to reduce T cell exhaustion with the disinhibition of early CART activation with CD5 deletion. To this goal, we used a clinically relevant sgRNA to knock out PD1 from CART19 cells and compared this to Mock and CD5 KO CART19 cells against CD19+ Nalm6 B-ALL cells in vivo. In a challenge dose model, PD1 KO CART19 were unable to enhance CART efficacy, while CD5 KO CART19 cells maintained strong tumor control that correlated with increased overall survival. We further compared CD5 versus PD1 KO CARTmeso in an in vivo solid tumor model of mesothelin+ PDAC. CD5 KO CARTmeso cells demonstrated enhanced tumor control and stronger response rates as determined by tumor volume and bioluminescence imaging. This increased tumor control allowed for enhanced survival that is likely explained by the significantly increased T cell count in CD5 KO CARTmeso-treated mice. Conclusion: In conclusion, this study demonstrates that CD5 is a negative regulator and possible novel immune checkpoint for adoptive T cell immunotherapies. We show that CD5 deletion leads to the enhancement of ACT anti-tumor function in several clinically relevant models of liquid and solid tumors.

UHRF1-Mediated Epigenetic Reprogramming Regulates Glycolysis to Promote Progression of B-Cell Acute Lymphoblastic Leukemia

Background Acute lymphoblastic leukemia (ALL) is a hematologic malignant disease with high heterogeneity, including B-ALL and T-ALL. The 5-year overall survival rate of children ALL has been significantly improved, which is over 90%. However, the prognosis of adult patients is not satisfactory, and the 5-year OS is only 30%-50%. Among them, the prognosis of patients with relapsed/refractory B-ALL is very poor. Exploring the molecular mechanism of disease progression is of great significance to improve the outcome. Methods Application of 10× Genomics chromium sequencer reveals the single-cell transcriptome profiling of peripheral blood or bone marrow MNC cells, which were from 6 patients with B-ALL（3 new diagnosed patients and 3 relapsed patients） and 3 healthy donors. The expression of key genes in 84 clinical blood or bone marrow samples was detected, and the prognosis was analyzed in TARGET database by bioinformatics technology. The molecular mechanism was investigated by RNAseq, 850k methylation microarray, qRT-PCR, Western blot, BSP, CHIP-qPCR and flow cytometry. Additionally, we identified a UHRF1 inhibitor using virtual compounds screening, and demonstrated its therapeutic efficacy both in vitro and vivo. Results Using Seurat method to cluster and dimension reduction of scRNA-seq data, 29 subclusters were obtained from 9 clinical samples. It mainly includes eight progenitor B cells, one B cell, six T cells, two NK cells, three monocytes, two neutrophils and three primordial erythrocytes. Nine samples were divided into three groups: Newly Diagnosis group (ND), Relapsed/Refractory group (RR) and Healthy Donors group (HD). The proportion of three subpopulations (cluster 2,5,16) in progenitor B cell group showed a decreasing trend of RR group > ND group > HD group. The marker genes of these three subgroups were enriched by GO and KEGG using ClusterProfiler R package. Cluster 2 was enriched into cancer signal pathway, cluster 5 was enriched into DNA replication process, and the marker genes of cluster 16 was mainly involved in metabolic pathway. The differential expression of these three subgroups between RR group and ND group was analyzed by FindMarkers function. The results showed that cluster 16 was the main differential cluster. The intergroup differential genes of cluster 16 mainly involve metabolic pathway, cancer pathway, cell cycle and p53 signaling pathway (Figure 1). The three groups were compared in pairs. After the intersection of differential genes in cluster 16 and all progenitor B cell groups, four common differential genes (HSPB1, UHRF1, H1F0 and IGFBP2) were screened. The results showed that the expression of UHRF1 gene was up-regulated in acute lymphoblastic leukemia patients, especially in patients with relapsed/refractory. Moreover, UHRF1 overexpression is an independent prognostic risk factor for B-ALL patients in our clinical data, and it is also a molecular marker of poor prognosis in the TARGET dataset. In vitro experiments, knockout of gene UHRF1 promoted the cycle arrest and apoptosis of B-ALL tumor cell line (Reh and nalm6), inhibited the proliferation of cells, and improved the drug sensitivity of vindesine. RNAseq and methylation microarray showed that the gene SFRP5 was demethylated and the expression of mRNA was overexpressed in UHRF1 knockout cell lines. This was verified by subsequent qRT-PCR, Western Blot and Bisulfite sequencing. Further studies showed that UHRF1 was involved in glycolysis by regulating the SFRP5-p38 MAPK-HK2 signal axis. Subsequently, through overexpressing SFRP5, we confirmed that upregulating SFRP5 can inhibit the P38 MAPK-HK2 signaling pathway, thereby suppressing glycolytic levels. Moreover, virtual compound screening identified UM164 as an inhibitor of UHRF1, which significantly inhibits P38 protein phosphorylation, downregulates HK2 expression, and reduces the production of glycolysis-related metabolites. Furthermore, UM164 demonstrated the ability to inhibit the proliferation of B-ALL cells both in vitro and in vivo, leading to prolonged survival in a B-ALL mouse model. In conclusion, we have unraveled the molecular mechanisms of epigenetic and metabolic rearrangements in relapsed/refractory B-ALL, and have provided potential targeted therapeutic strategies to improve its unfavorable prognosis.

Targeting Bone Marrow Microenvironment for Treatment of High-Risk B-Cell Acute Lymphoblastic Leukemia

Introduction B-cell acute lymphoblastic leukemia (B-ALL) is the most common form of cancer in children, accounting for approximately 20% of all pediatric cancers. Although treatment of B-ALL represents one of the success stories of modern medicine with 5-year survival rates approaching 90%, some children continue to have a dismal prognosis, particularly those with high-risk genetic alterations in their ALL cells or those who relapse. The leukemia microenvironment is an attractive novel therapeutic target, especially for children with high-risk leukemia as dose-limiting toxicities of conventional chemotherapeutic agents have prevented further improvements in survival. Aims In this study, we investigate whether therapeutically targeting the bone marrow (BM) osteoclasts can improve treatment outcome for B-ALL and the mechanisms of crosstalk between osteoclasts and leukemia cells. Methods We have established syngeneic and patient-derived xenograft (PDX) models for high-risk B- ALL that recapitulate the clinical symptoms of bone loss. For in vitro experiments, leukemia cells were cultured with osteoclast-derived conditioned media (CM) for 3 days. Results To evaluate the therapeutic potential of targeting osteoclasts during leukemia development, we administered zoledronic acid (2ug 5 days/week for 2 weeks), a bisphosphonate inhibitor of osteoclastic bone resorption, alone and in combination with imatinib (100mg/kg daily for 28 days) or dasatinib (10mg/kg daily for 28 days) in our BCR-ABL1 + B-ALL syngeneic mouse model. We showed that zoledronic acid treatment significantly reversed leukemia-induced bone loss by attenuating osteoclast activity. Zoledronic acid alone was found to enhance event-free survival in leukemia-bearing mice and further improvement was shown in combination with imatinib (p<0.05) or dasatinib (p<0.01). In addition, we administered zoledronic acid alone and in combination with vincristine, dexamethasone, and L-asparaginase (VXL) in a relapsed B-ALL PDX model. We further demonstrated that zoledronic acid alone or in combination with VXL improved survival compared to mice treated with vehicle (p<0.001) or VXL alone (p<0.01), respectively. In the clinical setting, we assessed the feasibility and safety of incorporating zoledronic acid early into leukemia therapy in three children with B-ALL. The patients were undergoing induction or consolidation chemotherapy and were administered zoledronic acid at the same time. Zoledronic acid was well tolerated without any significant adverse effects, indicating the feasibility of safely administering zoledronic acid in combination with chemotherapy in children with ALL. To gain mechanistic insight into the effect of osteoclasts on leukemia cells, we compared the molecular profile of the leukemia cells cultured with CM and control media using RNA sequencing. There were 30 differentially expressed genes identified at the 5% false discovery rate with a log fold-change value ≥1 or ≤-1. KEGG pathway analysis revealed a significant difference in cell cycle pathway in leukemia cells cultured with CM compared to control. Biological process analysis showed a positive regulation of B-cell proliferation and negative regulation of lymphocyte apoptotic process in leukemia cells cultured with CM compared to control. To functionally examine the effect of osteoclasts on leukemia cells, we showed that osteoclast-derived CM significantly increased B-ALL cell proliferation, as demonstrated by increased cell number (p<0.001), as well as reduced cell percentages in G0/G1 phases and increased S+G2/M phases (p<0.05) via cell cycle analyses. Using annexin V and DAPI staining, we further demonstrated that there were more live cells and less apoptotic and dead cells when the leukemia cells were cultured with CM compared to control (p<0.001). Together, our findings suggest that soluble factors produced by osteoclasts promote leukemia cell proliferation and survival. Conclusion Our study reveals osteoclasts play a role in leukemogenesis and leukemia-induced bone loss, and that targeting osteoclasts can provide dual clinical benefit in terms of bone health and improved survival for children with high-risk B-ALL.

Mitochondrial Isocitrate Dehydrogenase Inhibition Enhances CAR T-Cell Function By Restraining Antioxidant Metabolism and Histone Acetylation

Introduction: The efficacy of chimeric antigen receptor (CAR) T-cell therapy is hampered by relapse in hematologic malignancies and by hyporesponsiveness in solid tumors. Mitochondria are vital for the regulation of memory T-cell formation or exhaustion. A mitochondria-related compound screening was performed and we found that the FDA-approved isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib enhances long-lived memory CAR T-cell formation, and improves tumor clearance in vivo. IDH2 as the key component of the tricarboxylic acid (TCA) cycle, we hypothesized that IDH2 inhibition might promote CAR T-cell persistence by reprogramming the metabolism. The study aims to systematically evaluate the role and the mechanism of IDH2 inhibition on CAR T cells, and explore the application of enasidenib in CAR T-cell therapy. Methods and Results: To identify mitochondrial components that affect the long-term efficacy of CAR T-cells, we performed an in vitro screening using a mitochondria-related compound library based on the enrichment of the CD62L + CAR T-cell subset, which mainly contains T memory stem cells (T SCM) and central memory T cells (T CM). Among several candidate compounds that promoted memory CAR T-cell formation, enasidenib (ENA), an inhibitor of both wild-type and mutant IDH2 enzymes, was the most effective one. The proportion of CD62L has increased by over 20%. To determine the effect of ENA on exhaustion induced by tonic CAR signaling and tumor antigen stimulation, we measured the expression of inhibitory receptors PD-1, TIM-3, and LAG-3 and the level of apoptosis in freshly expanded CAR T cells and B-ALL encountered CAR T cells. CAR T cells treated with ENA exhibited reduced surface levels of those inhibitory receptors. We measured the production of granzyme B and IFNγ, and tumor rechallenge assay in CAR T cells after ENA treatment to assess the effector function, ENA treated CAR T cells exerted enhanced and sustained cytotoxicity in a tumor rechallenge assay at an extreme E:T ratio of 1:10. These results were observed not only in CD19-41BBζ CAR T cells, but also in CD19-28ζ and GD2-28ζ CAR T cells. CAR T cells expanded in the presence of ENA significantly prolonged survival of recipients after Nalm-6 infusion (p < 0.0001), extended in vivo ENA treatment further enhanced CAR T-cell expansion and tumor suppression. The median survival was extended from 45 to 67.5 days. Consistent with the effect of ENA treatment, IDH2 knockdown increased the proportions of the T scm/T n and T cm subsets, and alleviated CAR T-cell exhaustion. IDH2-knockdown CAR T cells had sustained cytotoxicity and higher CD62L expression after rounds of killing B-ALL cells in vitro. To systematically explore the effects of IDH2 inhibition on the metabolism of CAR T cells, relative metabolite amounts was measured using mass spectrometry (MS). IDH2-inhibited CAR T cells exhibited a substantial reduction of metabolites in the TCA cycle, including succinate, fumarate, and malate, and in the glycolysis pathway, including lactate, phosphoenolpyruvate. Mechanistically, IDH2 inhibition reprogram central carbon metabolism of CAR T cells by redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway. In addition, IDH2 limits cytosolic acetyl-CoA level to prevent histone acetylation that promotes memory cell formation. Significance: Our study indicates that metabolic intervention in CAR T cells with the FDA-approved IDH2 inhibitor enasidenib can advance current treatment, with better tumor eradication and CAR T-cell persistence.

Roles of Ribosomal S6 Kinases in Acute Leukemia and Normal Hematopoiesis

Ribosomal S6 kinases (RSKs) are serine-threonine kinases that are activated by the Ras/mitogen activated protein kinase signaling pathways and regulate cell proliferation and survival. There are 4 isoforms of RSK (RSK1, 2, 3, 4), and there is evidence that the isoforms mediate tissue-specific cellular functions. We hypothesized that RSK isoforms have distinct roles in hematopoiesisand evaluated RSK as a target for the treatment of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). To investigate isoform expression in hematopoiesis, we sorted cord blood mononuclear cells into hematopoietic progenitor stages and quantified RSK isoform mRNA by RT-qPCR. RSK1 expression remained consistent throughout hematopoiesis, whereas RSK2 expression decreased in the more differentiated populations. RSK3 was elevated in the lymphoid-primed multipotential progenitor and common myeloid progenitor stages and was greatly reduced in the more mature megakaryocyte-erythroid progenitor population. These results suggest that RSK proteins have isoform-specific roles in hematopoiesis. To analyze the importance of each isoform on hematopoiesis, we transduced cord blood CD34+ cells with lentiviral constructs expressing isoform-specific or control shRNAs. After liquid culture for 5 days, the cells were plated in methylcellulose media containing SCF, IL-3, GM-CSF, and EPO to determine the colony-forming potential. RSK1 and RSK2 knockdown decreased the colony-forming unit - erythroid colonies and increased the granulocyte/macrophage colony numbers. RSK3 knockdown increased the burst-forming unit - erythroid colonies and total colony numbers. After liquid culture days 8, 11, and 14, the erythroid and myeloid populations were analyzed by flow cytometry. RSK1 knockdown increased the CD11b+ and CD14+ myeloid populations and decreased the CD71+ erythroid population. RSK3 knockdown increased the erythroid population and decreased the myeloid populations. These data demonstrate that RSK1 and RSK3 have opposing effects on hematopoietic lineage determination. Overexpression of RSK proteins leads to aberrant cell survival and proliferation in many cancers including leukemia. We have previously reported that RSK and phosphorylated RSK are elevated in pediatric AML and are associated with a worse prognosis. There are only two orally bioavailable RSK inhibitors that have moved to phase I clinical trials: TAS0612 and PMD-026. TAS0612 is a multi-kinase inhibitor of RSK, AKT, and S6K. PMD-026 has high specificity to all four RSK isoforms. We evaluated the effects of both compounds in AML and ALL. To determine the effects of the RSK inhibitors on AML, we treated AML cell lines and patient samples with a range of doses of PMD-026, TAS0612, or 0.1% DMSO. After 3 days, we performed CellTiter Glo assays to determine the relative viable cell number and calculate the concentration of drug that inhibits cell growth by 50% (IC50). TAS0612 had an IC50 of 0.096-0.64 µM in AML cell lines Molm13, U937, AML193, and MV4;11. To test the cytotoxicity of TAS0612 on normal hematopoiesis, we plated healthy bone marrow mononuclear cells with TAS0612 in methylcellulose media. Colony formation was not adversely affected by TAS0612 up to 1 µM. PMD-026 was effective in Molm13, Kasumi-1, and MV4;11 with an IC50 of 0.34-0.55 µM and in AML patient samples 3123 and 4192 with an IC50 of 1.5-2.3 µM. PMD-026 did not affect healthy bone marrow colony formation up to 5 µM for a 2.5- to 10-fold therapeutic window. Furthermore, we tested the effects of RSK inhibitors in ALL. While PMD-026 was not effective, we found that B-ALL cell lines (REH, Nalm-6) and T-ALL cell lines (Loucy, Jurkat) were sensitive to RSK inhibition by TAS0612 with an IC50 of 0.056-0.19 µM. Western blot analysis of RSK protein revealed that RSK1, RSK2, and phospho-RSK proteins are elevated in ALL cell lines compared to healthy peripheral blood mononuclear cells. We are investigating the role of RSK and RSK isoforms in ALL. We found that RSK isoform expression is modified during hematopoiesis. Isoform expression influences early-fate decisions to differentiate into erythroid and myeloid lineages. RSK inhibitors PMD-026 and TAS0612 are effective at targeting AML and ALL cells with limited cytotoxicity to healthy cells, making them promising candidates for translation into clinical trials for AML and ALL.

Optogenetic Control of Oncogenic Signaling in B-Cell Malignancies

Background: B-cell receptor (BCR) signals are essential determinants of survival and proliferation throughout normal B-cell development. In B-cell malignancies, these signals are frequently generated by oncogenic mimics of the BCR signaling pathway. For instance, oncogenes in B-ALL, derived from B-cell precursors, typically mimic survival signals from a constitutively active pre-BCR, while tonic and chronic active BCR signaling were identified in mature B-cell lymphomas. Oncogenic BCR-signaling represents an important target of therapeutic intervention: for instance, small molecule inhibitors of SYK (e.g. entospletinib) and BTK (e.g. ibrutinib) have been developed to disrupt oncogenic BCR signaling in mature B-cell lymphomas. Besides SYK and BTK tyrosine kinases, oncogenic BCR-signaling leads to activation of PLCG2, which initiates the release of Ca 2+ from the ER into the cytoplasm. Thereby, calcium flux is decoded by NFATC1 or NF-kB based on fast (NFATC1) or slow (NF-kB) frequencies of Ca 2+-signals. Significance: To interrogate Ca 2+-signals as critical integration point of oncogenic BCR-signaling, we engineered cell lines and PDX with a GCaMP6s-biosensor, which allows tracing of calcium signaling in single cells over time ( Figure A). To cover B-cell malignancies from multiple stages of B-cell development, we engineered B-ALL (pro- and pre-B cells), mantle cell lymphoma (MCL, naïve B-cells), Burkitt's lymphoma (germinal center), DLBCL (post-GC), multiple myeloma (terminally differentiated plasma cells) and Hodgkin's disease (“crippled” BCR-deficient GC-B cells). Strikingly, B-cell malignancies exhibit autonomous Ca 2+-oscillations, which decreased in their frequency from 20 mHz (pro- and pre-B), 11 mHz (naïve), 4 mHz (germinal center) to 0 mHz in post-GC and terminally differentiated plasma cells based on the differentiation stage of their cell of origin ( Figure A). Results: Given the striking differences in oncogenic BCR-signaling as measured by autonomous Ca 2+ oscillations, we developed an optogenetic system to control the frequency and amplitude of Ca 2+ oscillations by blue light pulses ( Figure B). To model frequency-modulated Ca 2+ signaling in B-cells, we engineered murine pre-B cells carrying an optogenetic tool termed OptoCRAC, which enables reversible activation and inactivation of the plasma membrane Ca 2+ channel Orai1 by intermittent blue light irradiation. Time-lapse imaging of a fluorescent Ca 2+ reporter (R-CaMP1.07) confirmed rapid and reversible Ca 2+ influx in response to intermittent blue light pulses. To assess the phenotypic consequence of high-frequency Ca 2+ oscillations, we developed an LED-array-based optogenetic platform for cell culture plates. While low-frequency Ca 2+ oscillations (0.5 mHz) did not impact cell viability, high-frequency Ca 2+ oscillations (20 mHz) rapidly induced NFAT activation (5 min), and eventually cell death within 72 h ( Figure B). Mechanism and conclusions: To elucidate the mechanistic contribution of NFATC1 in B-cell death induced by fast Ca 2+ oscillations, we tested whether genetic deletion of Nfatc1 is sufficient to rescue B-cell death upon fast Ca 2+ oscillation induced by 20 mHz intermittent blue light pulses. As expected, while B-ALL cells retaining intact Nfatc1 rapidly decreased cell viability, the cells with Cre-mediated deletion persistently remained in cell culture upon 20 mHz Ca 2+ oscillation. In addition to genetic deletion of Nfatc1, we also found that inducible expression of mutant activators of NF-kB signaling were able to rescue B-cells from cell death induced by high-frequency oscillations: Concurrent expression of Card11 L232LI, MYD88 L265P and IKK2 S177E/S181E not only reduced the frequency of autonomous Ca 2+ oscillations but also enabled B-cell survival despite delivery of light-pulses and Ca 2+ oscillations at a fast 20 mHz frequency. We conclude that autonomous Ca 2+ oscillations represent a critical integration point of oncogenic BCR-signaling in B-cell malignancies and that optogenetic control of BCR-downstream Ca 2+ oscillations will reveal previously unrecognized vulnerabilities that can be exploited to increase efficacy of BCR-signaling inhibitors (e.g. ibrutinib, entospletinib, idelalisib) that are currently being used for the treatment of B-cell lymphomas.