B-ALL Cell Lines Research Articles

Targeting signaling pathways vulnerabilities for the treatment of IKZF1-deleted ph-negative B lymphoblastic leukemia.

7033 Background: Approximately 20% of children diagnosed with B lymphoblastic leukemia (B-ALL) will suffer a relapse, and most adults with B-ALL have a poor prognosis. Genome-wide association studies of B-ALL patients have identified frequent deletions of the gene IKZF1, encoding the master regulator of lymphoid development, IKAROS. These deletions are associated with therapy resistance, increased risk of relapse, and inferior survival. Currently, how loss of IKAROS function contributes to therapy resistance and increased risk of relapse is not fully understood. We used CRISPR-Cas9 genome editing to develop human B-ALL cell lines with various IKZF1 deletions that genetically and phenotypically recapitulate those occurring in patients. Using these isogenic cell lines, we have previously shown IKZF1 deletion results in cell-intrinsic chemoresistance and increased activation of the JAK/STAT signaling pathway (Rogers, Gupta et al. 2021). Methods: Given JAK/STAT is often dysregulated in poor-prognosis leukemia, we investigated the potential mechanisms of aberrant JAK/STAT activation and the therapeutic potential of targeting JAK/STAT in our engineered cell lines. We treated our cells with SH-4-54 (STAT3/5 inhibitor) or tofacitinib (JAK1 inhibitor) alone and in combination with dexamethasone. To elucidate how loss of IKAROS mediates an increase in JAK-STAT activity, we also performed RNAseq of known pathway regulators, comparing IKZF1 wild-type with IKZF1 knockout. Results: The JAK/STAT negative regulator Suppressor of Cytokine Signaling 2 (SOCS2) was significantly downregulated with IKZF1 deletion, validated by RTqPCR and immunoblotting. We further analyzed publicly-available RNAseq data from > 650 pediatric B-ALL samples, finding that SOCS2 expression is significantly lower in patients with low IKZF1expression (likely corresponding to IKZF1 deletion) compared to those with high IKZF1 expression. When we treated our engineered cell lines with tofacitinib or SH-4-54, IKZF1 wild-type cells were sensitive to each compound, suggesting JAK/STAT signaling plays a vital role in cell survival. In contrast, the IKZF1-deleted cells were relatively resistant to JAK/STAT inhibitors alone. However, in combination with dexamethasone, treatment of cells with sub-IC50 levels of SH-4-54 or tofacitinib resulted in re-sensitization to glucocorticoid-induced apoptosis. Conclusions: Our findings support that IKZF1 deletion leads to a targetable upregulation of the JAK/STAT pathway that, when inhibited, results in relative re-sensitization to dexamethasone. JAK/STAT pathway upregulation in IKZF1 deleted cells may be mediated by decreased expression of SOCS2. These results provide initial promise for targeting these vulnerabilities for the treatment of this poor-prognosis disease.

BCR::ABL1 tyrosine kinase inhibitors hamper the therapeutic efficacy of blinatumomab in vitro

PurposeAcute B-lymphoblastic leukemia (B-ALL) is a malignant disease characterized by accumulation of clonal immature lymphocytes in the bone marrow and peripheral blood. The approval of BCR::ABL1 tyrosine kinase inhibitors (TKI) such as imatinib, dasatinib, nilotinib and ponatinib marked a milestone in targeted therapy only for a subset of patients carrying the translocation t(9;22)(q34;q11). Immunotherapy with the bispecific antibody (bsAb) blinatumomab targeting CD19xCD3 revolutionized treatment of all B-ALL cases. The combination of both TKI and bsAb, so-called “dual targeting”, is currently under clinical investigation, although TKI might influence T cell effects.MethodsWe here investigated the combination of different TKI and blinatumomab in BCR::ABL1+ and BCR::ABL1− B-ALL cell lines and primary samples regarding T cell proliferation, differentiation, cytokine release and killing of tumor cells.ResultsIn vitro analysis revealed profound reduction of T cell proliferation, differentiation, cytokine release and killing of tumor cells upon application of BCR::ABL1 TKI with blinatumomab. Inhibition was more pronounced with dasatinib and ponatinib compared to nilotinib and imatinib. T cell signalling after CD3 stimulation was impaired by TKI mirrored by inhibition of LCK phosphorylation. This known off-target effect might influence the efficacy of bsAb therapy when combined with BCR::ABL1 TKI.ConclusionIn conclusion, we propose that nilotinib and imatinib might also be suitable substances for combination with blinatumomab and suggest evaluation in clinical trials.

In This Issue

See article, p. 95.Shree et al. find that up to a year after anti-CD20 treatment, most patients with lymphoma do not generate neutralizing antibody responses to COVID-19 vaccination. However, if patients are vaccinated and mount an antibody response prior to anti-CD20 treatment, they tend to maintain these antibody responses through subsequent anti-CD20 lymphoma therapy, suggesting a strategy of vaccinating first and treating later. These results also call to weigh the risks of maintenance therapy with rituximab or other B cell–depleting antibodies during the COVID-19 era.See article, p. 103.Downregulation of surface epitopes, including CD22, causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Zheng et al. report that CD22 mRNA is pervasively misspliced in B-ALL cell lines and primary samples. Some misspliced isoforms still encode plasma membrane–bound CD22 variants, while others lack the start codon in exon 2 and are noncoding. These exon 2–skipping isoforms are prevalent in high-risk B-ALL patients treated with the CD22-directed antibody–drug conjugate inotuzumab, where they correlate with low CD22 surface expression and are likely to account for treatment failure in two cases. Thus, aberrant CD22 splicing is an important mechanism of resistance to immunotherapy.See article, p. 116.Polycomb repressive epigenetic complexes are recurrently dysregulated in cancer. The mechanisms by which genetic alterations affect polycomb regulatory function to drive oncogenesis are poorly understood. By combining human genomics with biochemical and functional approaches, Schaefer et al. demonstrate that highly recurrent mutations in leukemia of the PRC1.1 subunits BCOR and BCORL1 dissociate DNA binding from enzymatic subunits. Uncoupling of the PRC1.1-repressive function from target genes alters epigenetic and signaling states, including FGFR derepression. BCOR deletion or truncation confers ibrutinib resistance, which is abrogated by FGFR inhibition. This epigenetic mechanism explains PRC1.1 tumor-suppressive activity and informs therapeutic approaches in PRC1.1-mutated cancer.See article, p. 136.IFNγ production has long been considered necessary for antitumor T-cell response. IFNγ also fuels macrophage activation in cytokine release syndrome (CRS), a significant toxicity of chimeric antigen receptor (CAR) T-cell therapy. Bailey et al. have discovered that either blockade or genetic knockout of IFNγ does not impair the anticancer activity of CAR T cells in preclinical models of hematologic malignancies. Further, there is reduced expression of immune checkpoint receptors on the CAR T cells and less activation of macrophages. Thus, it may be possible to separate the toxicity from the efficacy of CAR T cells for safer yet effective use.See article, p. 154.Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive, rare lymphoma of natural killer (NK) cell origin with very poor clinical outcomes. Mundy-Bosse et al. demonstrate that ENKTL cells are arrested at early stages of NK-cell maturation, characterized by broad epigenetic silencing of genes involved in normal NK-cell development. Treatment with the DNA hypomethylating agent 5-azacytidine led to a reduction of DNA hypermethylation, reexpression of normal NK-cell developmental genes, phenotypic NK-cell differentiation, and significant prolongation of survival in recipient mice. These studies lay the foundation for epigenetic-directed therapy targeting the developmental blockade in ENKTL.

Targeting Apoptosis Pathways With BCL2 and MDM2 Inhibitors in Adult B-cell Acute Lymphoblastic Leukemia.

In adult patients, the treatment outcome of acute lymphoblastic leukemia (ALL) remains suboptimal. Here, we used an ex vivo drug testing platform and comprehensive molecular profiling to discover new drug candidates for B-ALL. We analyzed sensitivity of 18 primary B-ALL adult patient samples to 64 drugs in a physiological concentration range. Whole-transcriptome sequencing and publicly available expression data were used to examine gene expression biomarkers for observed drug responses. Apoptotic modulators targeting BCL2 and MDM2 were highly effective. Philadelphia chromosome–negative (Ph–) samples were sensitive to both BCL2/BCL-W/BCL-XL-targeting agent navitoclax and BCL2-selective venetoclax, whereas Ph-positive (Ph+) samples were more sensitive to navitoclax. Expression of BCL2 was downregulated and BCL-W and BCL-XL upregulated in Ph+ ALL compared with Ph– samples, providing elucidation for the observed difference in drug responses. A majority of the samples were sensitive to MDM2 inhibitor idasanutlin. The regulatory protein MDM2 suppresses the function of tumor suppressor p53, leading to impaired apoptosis. In B-ALL, the expression of MDM2 was increased compared with other hematological malignancies. In B-ALL cell lines, a combination of BCL2 and MDM2 inhibitor was synergistic. In summary, antiapoptotic proteins including BCL2 and MDM2 comprise promising targets for future drug studies in B-ALL.

Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line.

The advent of CAR-T cell therapy has changed the face of clinical care for relapsed and refractory pre-B-acute lymphocytic leukemia (B-ALL) and lymphoma. Although curative responses are reported, long-term cures remain below 50%. Different CAR T-cell leukemia targets appear to have different mechanisms of CAR-T escape. For CD22, therapeutic evasion is linked to down-modulation of the number CD22 proteins expressed on the extracellular aspect of the leukemia cell plasma membrane. Recently, pharmacologic agents known to induce cellular differentiation or epigenetic modification of leukemia have been shown to impact CD22 and CD19 expression levels on B-ALL, and thereby increase sensitivity to CAR-T mediated cytolysis. We explored the impact of epigenetic modifiers and differentiation agents on leukemia cell lines of B cell origin, as well as normal B cells. We confirmed the activity of bryostatin to increase CD22 expression on model cell lines. However, bryostatin does not change CD22 levels on normal B cells. Furthermore, bryostatin inhibited CAR-T mediated cytolysis of the Raji Burkitt lymphoma cell line. Bryostatin increased the cytolysis by CD22 CAR-T for B-ALL cell lines by at least three mechanisms: 1) the previously reported increase in CD22 target cell numbers on the cell surface, 2) the induction of NK ligands, and 3) the induction of ligands that sensitize leukemia cells to activated T cell antigen-non-specific killing. The opposite effect was seen for Burkitt lymphoma, which arises from a more mature B cell lineage. These findings should caution investigators against a universal application of agents shown to increase killing of leukemia target cells by CAR-T in a specific disease class, and highlights that activation of non-CAR-mediated killing by activated T cells may play a significant role in the control of disease. We have termed the killing of leukemia targets, by a set of cell-surface receptors that does not overlap with NK-like killing “CTAK,” CAR-T Cell antigen-non-specific killing.

Cytofluorimetric assay to investigate variability in blinatumomab in vitro response.

The T-cell engager antibody blinatumomab (BlincytoT⁢M) represents a promising rescue therapy for relapsed/refractory CD19+ acute lymphoblastic leukemia (B-ALL), although ~20-30% of patients still do not respond to treatment. Blinatumomab creates a tight synapsis between CD3+ T-lymphocytes and leukemic CD19+ B-cells, resulting in a granzyme B (GzB)-mediated specific lysis of leukemic cells. Aim of the study was to provide evidence that variability in blinatumomab response could have a genetic basis in PAX5, one of the most often mutated genes in B-ALL, affecting the CD19 surface expression on lymphoblasts, and could be explored in vitro by means of a cytofluorimetric assay, staining both surface antigens (CD45, CD19 and CD3) and intracytoplasmic markers (7AAD, Syto16). Two human immortalized B-ALL cell lines (NALM6 and REH) were chosen for their different PAX5 and CD19 protein levels, as verified by western blot and flow cytometry, respectively. In contrast to NALM6, REH cells do not express the full-length PAX5 protein and show less CD19 on the cell surface (fluorescence peak median intensity: 9155 versus 28895). Co-cultures of CD3+ T-lymphocytes from healthy donors and B-ALL cell lines were seeded at an effector-to-target cell ratio of 1:10 for simulating the condition existing in the bone marrow due to the malignant invasion of blast cells. Co-cultures were exposed in vitro to blinatumomab and the simultaneous increase in blast mortality and T-lymphocytes activation induced by the drug was observed at day +7 (both effects: p < 0.0001 versus untreated, two-way ANOVA, Bonferroni post-test), and was particularly pronounced in REH compared to NALM6 co-cultures (p < 0.05). Surprisingly, daily release of GzB in supernatants, measured by an ELISA assay, was significantly lower in drug-exposed REH co-cultures compared to NALM6 at early time-points (days +3 and +4, p-value < 0.0001, three-way ANOVA), reaching a comparable plateau only towards the end of the incubation period (at day +5). Only 2 out of 5 primary co-cultures of leukemic and mononuclear cells isolated from bone marrow aspirates of B-ALL patients (age: median 10.7 years, interquartile range (IQR) 3.4; males: 60%) responded to the drug in vitro (simultaneous blast mortality and T-lymphocyte activation: both effects: p < 0.0001 versus untreated) and at different drug concentrations. Results were unrelated to the percentages of immature CD19+ B-cells in the diagnostic samples. In conclusion, cytofluorimetric analysis can highlight the different response induced by blinatumomab among co-cultures. Whether and how this difference is affected by PAX5-regulated CD19 expression is unclear and whether it is predictive of in vivo response to therapy remains to be established. Further dedicated studies are required to investigate these issues in detail.

Characterization of a novel glucocorticoid-resistant human B-cell acute lymphoblastic leukemia cell line, with AMPK, mTOR and fatty acid synthesis pathway inhibition

BackgroundAcquired glucocorticoid (GC) resistance remains the main obstacle in acute lymphoblastic leukemia (ALL) therapy. The aim of the present study was to establish a novel GC-resistant B-ALL cell line and investigate its biological characteristics.MethodsA cell culture technique was used to establish the GC-resistant cell line from the parental cell, NALM-6. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, DNA fingerprinting analysis and whole transcriptome sequencing (WTS) were used to characterize the GC-resistant cell lines. Nude mice were used for xenograft studies.ResultsThe GC-resistant cell line, NALM-6/HDR, was established by culturing NALM-6 cells under hypoxia for 5 weeks with a single dexamethasone (Dex) treatment. We subcloned the NALM-6/HDR cell lines, and got 6 monoclone Dex-resistant cell lines, NALM-6/HDR-C1, C3, C4, C5, C6 and C9 with resistance index (RI) ranging from 20,000–50,000. NALM-6/HDR and its monoclone cell line, NALM-6/HDR-C5, exhibited moderate (RI 5–15) to high resistance (RI > 20) to Ara-c; low or no cross-resistance to L-Asp, VCR, DNR, and MTX (RI < 5). STR analysis confirmed that NALM-6/HDR and NALM-6/H were all derived from NALM-6. All these cells derived from NALM-6 showed similar morphology, growth curves, immunophenotype, chromosomal karyotype and tumorigenicity. WTS analysis revealed that the main metabolic differences between NALM-6 or NALM-6/H (GC-sensitive) and NALM-6/HDR (GC-resistant) were lipid and carbohydrates metabolism. Western blotting analysis showed that NALM-6/HDR cells had a low expression of GR and p-GR. Moreover, AMPK, mTORC1, glycolysis and de novo fatty acid synthesis (FAS) pathway were inhibited in NALM-6/HDR when compared with NALM-6.ConclusionsNALM-6/HDR cell line may represent a subtype of B-ALL cells in patients who acquired GC and Ara-c resistance during the treatment. These patients may get little benefit from the available therapy target of AMPK, mTORC1, glycolysis and FAS pathway.

Priming Death Receptor Mediated Apoptosis with Arginine Starvation Sensitises Arginine Auxotrophic B-ALL to CAR-T

BackgroundChimeric antigen receptor (CAR)-T cell therapy has revolutionised the treatment of relapsed or refractory B-ALL in children and young adults with unprecedented response rates. However, primary resistance and relapse are unresolved challenges that limit long term benefit in a significant proportion of patients. Death receptor mediated extrinsic apoptosis is a key component of CAR-T cytotoxicity and impairment of this system, of which CAR-T derived TRAIL (tumour necrosis factor related apoptosis inducing ligand) is a key initiator, is a principal driver of primary resistance. Arginine deprivation with the therapeutic enzyme ADI-PEG20 (pegylated arginine deiminase) sensitises cancers deficient in the enzyme argininosuccinate synthase (ASS1) to the apoptosis initiating activity of TRAIL through tumour cell surface upregulation of death receptors DR4 and DR5. Whether this effect could potentiate the TRAIL-DR activity in CAR-T therapy has not been explored.AimWe tested the hypothesis that ADI-PEG20 treatment can sensitise susceptible B-ALL to anti-CD19 CAR-T through priming of death receptor mediated apoptosis signalling.MethodsThe effect of ADI-PEG20 on cell survival and death receptor expression in B-ALL cell lines and primary samples was analysed by flow cytometry. Second generation anti-CD19 CAR-T cells with a CD28 costimulatory domain were generated by retroviral transduction of activated peripheral blood mononuclear cells (PBMC) from healthy donors. For CAR-T co-culture experiments, B-ALL cell lines were pre-treated with ADI-PEG20 before washing and re-suspending in arginine replete media prior to CAR-T cell addition.ResultsTo establish potential susceptibility of B-ALL to ADI-PEG20 we measured expression of ASS1, which inversely correlates with sensitivity to the drug, using combined in situ immunohistochemistry (n=6) and RT-qPCR (n=7). ASS1 deficiency was consistently seen in this series of primary samples suggesting the potential utility of ADI-PEG20 in B-ALL, with comparable expression levels to those seen in a cohort of primary AML samples proven to be sensitive to the drug (figure 1a).Next, to examine variation in ASS1 expression between genetically defined subtypes of B-ALL we re-analysed transcriptome data from a cohort of 215 patients treated on the ECOG E2993 trial, filtered into a network of 58 genes generated according to known or predicted interaction with ASS1. We found an enrichment of Philadelphia chromosome positive (Ph+) and Philadelphia-like (Ph-L) samples in the cluster characterised by lowest ASS1 expression along with high HIF1A expression, matching a recurrent pattern reported in other ADI-PEG20 sensitive tumours. This therefore predicts that among B-ALL subtypes, Ph+ and Ph-L are likely to be most sensitive to therapeutic arginine deprivation.We then functionally confirmed, using in vitro cell line (n=3) and in vivo patient derived xenograft models of B-ALL (n=2), that ASS1 deficiency is required for ADI-PEG20 sensitivity. Using the ASS1-low, Ph-L cell line MUTZ-5, we established that ADI-PEG20 induced apoptosis accompanies cell surface upregulation of both DR4 and DR5 expression. Upregulation of DR4 was observed to follow an upwards trend after treated cells were washed and re-suspended in arginine replete media, suggesting that transient arginine starvation can commit ASS1-low B-ALL to a state of apoptotic priming (figure 1b).With confirmed engagement of arginine starvation and death receptor upregulation we tested the synergy potential of ADI-PEG20 pre-treatment of MUTZ-5 followed by CAR-T, utilising calculated combination drug indices (CDIs). Across independent PBMC donors (n=3) we observed greater potency killing of CD19 + leukaemia cells in the combination treated co-cultures when compared to the single agent treated conditions, with CDIs consistently less than 1 confirming a synergistic effect (figure 1c).ConclusionOur study proposes a synergistic interaction between the arginine depleting enzyme ADI-PEG20 and anti-CD19 CAR-T for the treatment of ASS1 deficient B-ALL, whereby priming of death receptor signalling may underlie enhanced CAR-T cytotoxicity against CD19 + tumour cells. These data support an emerging framework for CAR-T optimisation based on targeting of the death receptor mediated extrinsic apoptosis pathway and can inform future refinements in the development of cellular immunotherapy. [Display omitted] DisclosuresBomalaski: Polaris Pharmaceuticals Inc.: Current Employment. Maher: Leucid Bio: Other: Chief Scientific Officer. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

Synergism between IGF2BP1 and ETV6-RUNX1 in the Pathogenesis of ETV6-RUNX1 Positive B-Acute Lymphoblastic Leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common pediatric haematological malignancy with a prevalence of around 85% cases of B cell origin. ETV6-RUNX1 is the most common genetic aberration in childhood B-ALL with a prevalence of around 25% in Western countries and a lower prevalence in Asian countries. ETV6-RUNX1 presence portends a good prognosis in B-ALL. ETV6-RUNX1 translocation has been observed in 1-5% of healthy newborns, indicating that it is not sufficient for leukemogenesis, and the need to accumulate secondary mutations for leukemogenesis.We have previously demonstrated the specific overexpression of Insulin like Growth Factor 2 Binding Protein 1 (IGF2BP1), an oncofetal RNA binding protein, in ETV6-RUNX1 positive B-ALL. Overexpression of the ETV6-RUNX1 fusion protein in 7OZ/3, a murine B-ALL cell line increased endogenous Igf2bp1 levels in a dose dependent manner.Conversely, knockout of IGF2BP1 in Reh-Cas9-GFP expressing B-ALL cell line, a modified version of Reh (cell line positive for ETV6-RUNX1 translocation), using 2 different guide RNAs led to a decrease in the ETV6-RUNX1 transcript expression in the IGF2BP1 KO Reh-Cas9 cells. This was accompanied by a decrease in cell proliferation and increased chemosensitivity to prednisolone.A similar phenotype was observed in Reh-Cas9 cells after knockout of ETV6-RUNX1 fusion transcript hinting towards a synergism between the two proteins in tumour cell survival and chemosensitivity. Interestingly, there appears to be a positive correlation between prednisolone resistance and ETV6-RUNX1 positivity in our patient cohort too, thus supporting the in vitro finding. Previous studies have shown the binding of IGF2BP1 to the ETV6-RUNX1 fusion transcript which was also confirmed by us.To study the in vivo synergistic effect between ETV6-RUNX1 fusion protein and IGF2BP1, we used a murine bone marrow transplant model. The individual genes were overexpressed alone (IGF2BP1/ETV6-RUNX1) or together (IGF2BP1+ETV6-RUNX1) in separate groups of lethally irradiated mice (n=8 per group). In the group where both the genes were overexpressed together, there was clonal expansion and hypercellularity in the bone marrow. This was accompanied by an increase in the number of lineage negative cells (Lin-), progenitors including HSCs, LMPPs and c-kit+ cells which also showed high Ki67 positivity. The marrow hypercellularity led to extramedullary haematopoiesis which was seen as significant splenomegaly.In addition, analysis of the endogenous levels of Igf2bp1 in the bone marrow of the primary murine samples belonging to the ETV6-RUNX1 fusion protein overexpression group resulted in an increase in its levels, thus confirming our in vitro finding suggesting its induction by ETV6-RUNX1 fusion protein.RNA-Seq of IGF2BP1 KO cells identified enrichment of targets of the TNF alpha/NFκB signalling and K-Ras pathways hinting towards their dependency on IGF2BP1. The direct targets of IGF2BP1 were identified by RIP-Seq (RNA-Immunoprecipitation-Seq) analysis in Reh cell line. Numerous genes from the oncogenic signalling pathways including the TNF alpha/ NFκB signalling pathway were identified as IGF2BP1 targets. We validated some overexpressed genes from the TNF alpha/NFκB signalling and PI3K pathway in primary patient samples. We found significant overexpression of some of the genes (IL6ST, NFAT5, CDK6, MDM2, CCND1, NGFR) belonging to these pathways in the ETV6-RUNX1 positive group (n=39) compared to the negative cohort (n=111) as well as MACS sorted CD19+ B-cells (n=5). The decrease in the ETV6-RUNX1 transcript levels after downregulation of IGF2BP1 and overlap between the oncogenic pathways clearly shows an interdependency between the two genes.Overall, our results suggest a potential feedback mechanism between ETV6-RUNX1 fusion protein and IGF2BP1 in the pathogenesis of leukemia development and IGF2BP1 can be utilised as an ideal therapeutic target for this particular subtype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ikaros Mediates Antigen Escape Following CD19 CAR T Cell Therapy in r/r B-ALL

Despite impressive response to front-line treatment, relapsed or refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) remains one of the most common causes of death in children with cancer. In pediatric and adult r/r B-ALL, CD19 CAR T cell (CART19) therapy has shown impressive results with over 80% of patients achieving complete remission. However, as an unintended consequence of long-term persistence of CAR T cells and the resulting sustained pressure against CD19 expressing cells, 30 - 70% of initial responders will eventually relapse with CD19 loss. Early identification of patients that will respond successfully to CART19 therapy vs. those at risk for CD19 Neg relapse would support clinical decision making and improve patient care.To address this problem, we profiled by mass cytometry (CyTOF) 25 pre-CART19 samples including patients that achieved a durable complete remission (CR, n = 6), patients that underwent CD19 Neg (n = 11) or CD19 Pos relapse (n = 4) and patients that were refractory to CART19 (n = 4). For 14 of those patients, we also analyzed paired post-CART19 samples: 8 from CD19 Neg relapse, 2 from CD19 Pos relapse, and 4 from refractory tumors. We observed a significant increase in the proportion of leukemic early progenitor non-B cells (called early-non-BI) after CD19 Neg relapse, suggesting that CD19 loss is associated with the loss of other B cell features. In addition, in the pre-CART19 samples we identified a subpopulation of CD19 Pos pro-B cells that resemble early-non-BI cells. We hypothesized that these pro-B “discordant” cells are able to lose CD19 expression and escape the immune pressure exerted by the CART19, leading to CD19 Neg relapse. Closer analysis revealed lower levels of IKAROS in pro-B discordant cells from patients that underwent CD19 Neg relapse, than in those that achieved a durable CR.To further explore the difference between pro-B discordant cells from patients that achieved a durable CR or underwent CD19 Neg relapse, we performed single cell REAP-seq (RNA expression and protein sequencing) from 3 pre-CART19 CR and 3 pre-CART19 CD19 Neg relapse r/r B-ALL samples. From this analysis, we confirmed a subpopulation of leukemic pro-B like cells with decreased expression of IKZF1 in those patients that underwent CD19 Neg relapse.IKAROS is a transcription factor that plays a crucial role in B cell lineage specification and commitment and is a known B-ALL tumor suppressor. Genetic alterations affecting the IKAROS gene, IKZF1, have been associated with poor response to front-line treatment and they are a factor in relapse risk stratification. However, a role for IKAROS in CART19 failure has not been described. To assess if there is a correlation between the levels of IKAROS and CD19 surface expression, we manipulated IKAROS levels in B-ALL cell lines through RNA silencing and targeted protein degradation approaches. Consistently, we observed that CD19 surface expression diminishes upon IKAROS downregulation. To determine how IKAROS regulates CD19 expression, we performed CyTOF, RNA-seq and ATAC-seq analysis of isogenic B-ALL cell lines with wild-type or reduced (IKAROS KD) IKAROS levels. While the chromatin accessibility to CD19 regulatory regions did not change, CD19 mRNA levels were lower in IKAROS KD cells. Interestingly, we found that IKAROS regulates several genes associated with mRNA splicing. In particular, CD19 mRNA preferentially retains intron 10 in IKAROS KD cells. The inclusion of intron 10 introduces several premature stop codons. Moreover, IKAROS KD B-ALL cells more closely resemble myeloid cells based on their gene expression profiles, suggesting a loss in their B cell identity.In summary, this is the first description of a leukemia-specific subpopulation of CD19 Pos pro-B discordant cells in patients prior CART19 administration. Furthermore, lower levels of IKAROS in this subpopulation correlates with poor response to CART19 therapy. Preliminary molecular analysis suggests that low IKAROS result in retention of CD19 intron 10 and loss of B-cell identity (Figure 1). Future research will focus on in-depth characterization of the myeloid-like phenotype induced by low levels of IKAROS expression, and the impact that intron 10 retention has on CD19 mRNA stability and surface expression. [Display omitted] DisclosuresMullighan: Pfizer: Research Funding; Amgen: Current equity holder in publicly-traded company; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Barrett: Tmunity Therapeutics: Current Employment. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

HDACi Targets IKZF1 Deletion High-Risk Acute Lymphoblastic Leukemia By Inducing IKZF1 Expression and Rescuing IKZF1 Function in Vitro and In Vivo

The IKZF1 gene codes the transcription factor IKAROS with key regulatory functions in lymphopoiesis. Recurrent focal IKZF1 deletions (IKZF1 del), which affects the coding regions of IKZF1, have been identified as poor outcome in 30-40% adult B-ALL. Loss of IKAROS confer stem cell-like phenotype, upregulation of self-renewal capacity and cell-adhesion molecules, and TKI resistance. However, there are not specifically therapeutic options for IKZF1 del ALL and current protocols could not abrogate the adverse effect of IKZF1 del.Considering that IKAROS acts as a key component of the nucleosome remodeling and deacetylation (NuRD) complex engaging in development and metabolism, we speculate that epigenetic drugs, such as HDACi, might play a potent role in IKZF1 del high-risk B-ALL.Firstly, B-ALL cell lines (IKZF1 del: MUTZ-5, MHH-CALL-4; IKZF1 wt: NALM6) and primary patient samples (n=10, 5 with IKZF1 del and 5 with IKZF1 wt) were treated with different HDACi, including valproic acid, vorinostat, romidepsin, RGFP966 and a novel HDAC-selective inhibitor tucidinostat. But noteworthily, only tucidinostat yielded specific and selective proliferation inhibition in IKZF1 del cell line(IC 50=1.377±0.05) and IKZF1 del patients samples (IC 50=2.318±0.07), compared with the effect on IKZF1 wt cells. Interestingly, tucidinostat induced remarked increase of mRNA and protein of IKZF1 expression in leukemia bulk and IKZF1 del single cell. Seahorse metabolic flux assay, lactate and ATP measurements was performed and revealed that tucidinostat treatment reduced glycolysis (P=0.0067), lactic acid (P＜0.0001) and ATP level (P＜0.0001) in IKZF1 del B-ALL cell lines. To verify metabolic change is depend on IKZF1 induction or not,dominant-negative Ikaros isoform 6 (DN-IK6), deletion of exons 4-7, was transfected into IKZF1 wt Nalm-6 cell line to negative regulate of IKZF1 wide-type expression. Overexpression of DN-IK6 in Nalm-6, increases sensitivity to tucidinostat, glycolytic capacity(p=0.05) and glycolytic reserve (p=0.012) also increases. While tucidinostat treating with the IK6-Nalm-6, tucidinostat would restore the transcriptional repressor function of the remaining wild-type IKZF1 allele and decrease glycolytic capacity(p=0.011) and glycolytic reserve(p=0.014). Notably, the metabolic rate-limiting enzymes HK2 and PKM2 were strongly repressed. These data indicate that tucidinostat reverses the metabolic reprogramming of glycolysis or Warburg effect in IKZF1 del B-ALL in an IKZF1-inducing dependent manner. For in vivo study, PDX model with immunodeficient NOD/SCID/IL2Rgnull mice were injected with heavily-treated refractory/relapsed IKZF1 del B-ALL patient samples (n=2) and treated with tucidinostat with different dosage of 5-12.5mg/kg/day. Administration of tucidinostat observed IKAROS expression trajectory and resulted in prolonged animal survival in IKZF1 del B-ALL PDX model（P＜0.0001). Secondary transplantation of ALL cells from tucidinostat or vehicle-treated (1 x10 6) recipients revealed significantly improved survival in tucidinostat -treated group (p= 0.0235). These results indicate that tucidinostat treatment might elimination leukemia-initiating cells.Additionally, to profile the IKZF1 del B-ALL chromatin accessibility changes after tucidinostat-treatment. We performed ATAC-seq and observed a clear increase in accessibility at TCA cycle related gene and decrease in accessibility at glycolysis related gene.Furthermore, tucidinostat, formerly known as chidamide, was added to an open-label, one-arm PDT-Ph-like-ALL trial targeting adult Ph-like ALL, which is characterized with high frequency in IKZF1 deletions (Clinicaltrials.gov. NCT03564470). Preliminary data of PDT-Ph-like-ALL indicate that tucidinostat was effective and well-tolerated, yielded promising response in IKZF1 del Ph-like ALL (ASH2018, poster 4011; EHA 2019, PF181).Collectively, our study demonstrates that the novel HDAC-selective inhibitor, tucidinostat, could specifically target IKZF1 del high-risk B-ALL, by restoring the IKZF1 expression, resulting in attenuation of proliferation, reverse the Warburg effect and improvement of the survival in PDX model and preliminary data in clinical trial. These findings provide mechanistic insights and a promising therapeutic strategy for IKZF1 haploinsufficiency alterations B-ALL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Using NK-Derived Exosomes to Treat Leukemia

Natural killer cells (NK) are components of the innate immune system which play an important role in surveillance of cancer cells. Despite promising results in clinical trials, the use of NK-based therapies is limited due to safety issues. Exosomes are nanosized extracellular vesicles secreted by all types of cells. In recent years, exosomes have emerged as a powerful, natural therapeutic tool due to their low immunogenicity, low cytotoxicity, prolonged bioavailability and superior targeting ability. Since exosomes are known to carry cargo that reflects their cell of origin, we were prompted to test whether NK-derived exosomes (NK exo) maintain the anti-leukemia capacity of NK cells. To test the feasibility of this approach we undertook a set of preliminary experiments and evaluated the anti-leukemic potential of NK exo in-vitro on a wide variety of leukemic cell lines and patient-derived samples while also striving to assess their killing potential in-vivo using a murine xenograft model . This study aims to provide the pre-clinical evidence needed to test the NKexo approach in future clinical studies with the intention of ultimately developing an acellular “off-the-shelf” product to treat leukemia.Initially, we isolated NK exo from the engineered NK92MI cell line by ultracentrifugation and demonstrated the presence of large amounts of 100-200nm cap-shaped particles by means of nano-tracking-analysis and transmission-electron-microscopy. We then showed that these particles express the exosomal-characteristic CD63- and CD81- and NK-characteristic CD56-biomarkers. Next, we evaluated the effect of NK exo on CML (K562), T-ALL (Jurkat), B-ALL (UoC) and AML (HL-60 and KG1a) cell lines. NK exo treatment initiated a time- and dose-dependent reduction in viability and induction of apoptosis in all cell lines tested. The effect of NK exo exposure on cell viability and apoptosis was reduced in the presence of the pan-caspase inhibitor Z-VAD-FMK suggesting that NK exo exert their cytotoxic effect in a caspase-dependent manner. In addition, exposure to NK exo led to a significant increase in apoptosis in treatment naive AML, CLL and ALL patient-derived mononuclear cells. Moreover, we witnessed a significant reduction in colony formation of mononuclear cells harvested from treatment naive CML and AML patients in the presence of NK exo. While leukemia cells were targeted and severely affected by NK exo, healthy B-cells remained unaffected, indicating a selective effect. The selective trait of NK exo was also confirmed via an exosome uptake assay which demonstrated that NK exo were taken up specifically by leukemic cells but not by healthy B-cells.In an in-vivo xenograft model for AML; NOD-SCID IL2Rgama null (NSG) mice were engrafted with the well characterized human AML cell line HL60, and were then intravenously injected with NK exo. Our preliminary results indicate that NK exo treatment improves overall mice survival indicating an anti-leukemic effect of NK exo in-vivo.In this “proof-of-concept” study, we show that NK exo, similar to their cell-of-origin, have a strong and selective anti-leukemia effect. To provide the pre-clinical evidence needed to test the NK exo approach in future clinical studies, we are currently continuing to test the anti-leukemic potential of NK exo in-vivo on a xenograft murine model. Our preliminary results propose the use of NK exo as a novel therapeutic platform to combat leukemia. More broadly, since NK cells target a wide range of cancer cells, not just leukemia cells, and since we have shown that NK exo maintain the anti-leukemic capacity of their donor cells, this approach may become applicable in other hematological and solid malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Methionine Restriction As a Novel Therapeutic Strategy for MLL (KMT2A)-Rearranged Acute Lymphoblastic Leukemia

Background:MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is a rare but aggressive subset that represents 5% of childhood ALL cases, and accounts for about 70% of infant leukemias. While overall survival in these young children is around 50%, after relapse, MLLr ALL becomes an almost incurable disease, highlighting the urgent clinical need for new strategies for this patient group. The histone methyl transferase function of the MLL fusion protein complex requires the methionine metabolite s-adenosylmethionine (SAM) as methyl donor, suggesting a selective sensitivity of MLL-r ALL for perturbations in methionine availability. Recent studies in solid tumor models suggest clinical utility of methionine restricted diets or oral administration of methionine depleting enzyme Methionine Gamma Lyase (MGL) to be safe and effective. Therefore, we explored the effect of methionine restriction (MR) as a potential, new therapy for MLLr ALL.Methods:We compared the effects of MR on metabolic activity and viability between MLLr and non-MLLr pre-BCP ALL cell lines using enzymatic depletion, small molecule inhibitors targeting methionine metabolism, and restrictive culture conditions. To identify intrinsic metabolic differences between MLLr and non-MLLr cells and explore how MR impinges on their metabolic state, we performed global metabolomics on MLLr SEM cells and non-MLLr NALM6 cells cultured with complete depletion of methionine. Additionally, we used RNA sequencing to assess the global effects of MR on gene expression, and a CRISPR/Cas9-based reverse genetic screen to identify sensitizers towards MR. Results were validated in vitro using targeted knockouts and small-molecule inhibitors, as well as in vivo using a 95% methionine restricted diet. Immunocompromised mice were engrafted with MLLr SEM cells and 7 days after transplantation, mice were randomized to control or 95% MR diet. Leukemia progression was monitored by flowcytometric detection of human lymphocytes in the blood.Results:We observed that depletion of methionine reduces metabolic activity in almost all BCP-ALL (B-ALL) cell lines, however, only in MLLr B-ALL cell lines was rapid apoptosis induced (Figure 1A). Global metabolic profiling revealed significant basal metabolic differences, of note being SAM, whose levels were approximately 5-fold higher in MLLr SEM cells compared to non-MLLr NALM6 cells. Consistent with this, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells (Figure 1A). Metabolomic profiling also highlighted different salvage mechanisms at play in NALM6 cells, with the folate cycle and polyamine synthesis pathway being activated upon MR. Together, these results indicate that MLLr B-ALL cells are selectively sensitive to MR. In line with this, RNASeq data showed significant decreased expression of several known MLL fusion target genes such as PROM1, HOXA10, and MEIS1 in response to MR.To obtain further insight into the pathways involved in the response to MR and to identify potential therapeutic targets that further sensitize cells to MR, we performed a CRISPR/Cas9-based screen. This identified three members of the Bromodomain- and extra-terminal domain (BET) family as potential modifiers of the response to MR in SEM cells. Indeed, RNAseq analysis showed that Myc activity as a proxy of BRD4 function, was strongly suppressed by MR. Finally, preliminary results show the efficacy of dietary intervention alone on leukemia progression. We observe with 95% MR diet, significant delays on leukemic growth (Figure 1B). Moreover, the MR diet was well tolerated, as indicated by minimal weight loss after two months. Although further studies are needed, we anticipate that targeting epigenetic regulators or use of conventional therapies in combination with MR would further potentiate this effect.Conclusions:MLLr leukemic cells have an increased dependency on S-adenosylmethionine and therefore show increased vulnerability to methionine depletion. Limiting methionine availability, either by enzymatic methionine depletion or dietary restriction could provide a novel therapeutic option for this patient group, particularly when combined with other therapies. The availability of an FDA approved methionine-free formula facilitates rapid translation to clinical practice, particularly in infants. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterizing the Mechanism of NE and P3 Cross-Presentation in B Cell-Acute Lymphoblastic Leukemia

IntroductionPR1 is a nonameric HLA-A2 restricted leukemia-associated antigen derived from serine proteases neutrophil elastase (NE) and proteinase 3 (P3). NE and P3 are primarily expressed in cells of myeloid lineage including granulocytes, bone marrow progenitors and myeloid leukemia. However, NE and P3 are also cross-presented by antigen presenting cells (APCs) such as B-cells and some non-hematopoietic solid tumors, a mechanism whereby exogenous antigens are endocytosed and presented on HLA class I molecules. Therefore, identifying non-myeloid tumors capable of PR1 cross-presentation broadens the application of PR1-targeted immunotherapies, which to date include PR1 peptide vaccine, PR1 cellular therapy, and 8F4, a T cell receptor (TCR)-like monoclonal antibody (mAb).One possible source of NE and P3 in the microenvironment is neutrophil extracellular traps (NETs). NETs are composed of deoxyribonucleic acid (DNA)/histones and the antimicrobial proteases, NE and P3, which are extruded from activated polymorphonuclear neutrophils (PMNs) during inflammation. Although a main function of NETs is elimination of pathogens, studies have shown immune-regulatory effects of NETs. Thus there is a great interest in understanding the role of NETs in modulating adaptive immune system in the setting of anti-tumor immunity. We hypothesized that NETs facilitate NE and P3 cross-presentation in malignant B-cell acute lymphoblastic leukemia (ALL) due to their high abundance in the bone marrow, which could lead to susceptibility to PR1-targeted immunotherapies.MethodsB-ALL cell lines SB, Nalm6, RS4 and Nalm6 were co-cultured with soluble NE and P3 (10 ug/mL) and PMNs at a ratio of 3:1 (PMN: ALL) in low serum media and analyzed for NE and P3 cross-presentation by staining for HLA-A2/PR1 complex using 8F4 mAb. T2 cells pulsed with PR1 and HIV peptide were used as a positive and negative control, respectively. To determine susceptibility to killing my PR1-CTLs, SB cell line was co-cultured with 10 μg/mL soluble NE and P3 for 24 hours and incubated with healthy PR1-CTLs for 4 hours following a protocol for a standard calcein-AM cytotoxicity assay. T2 cells were pulsed with PR1 and CG1 peptide as a positive and negative control, respectively.To identify NETs as a source of NE and P3, healthy PMNs were treated with ionomycin to induce NETosis, co-cultured with B-ALL cell lines at a ratio of 3:1 (PMN: ALL) overnight and analyzed for NE and P3 uptake. NETs were also treated with DNAse to determine the effect on uptake.ResultsThe B-ALL cell line, SB, cross-presented PR1 from soluble P3 and NE, as well as supernatants derived from quiescent PMNs. There was a 1.3 fold significant increase in PR1/HLA-A2 surface expression when co-cultured with soluble and PMN-associated protease (Figure 1A). We then investigated whether PR1 cross-presentation by SB cell line rendered it susceptible to killing by PR1-CTLs. Dose-dependent killing of SB cells was observed following PR1 cross-presentation compared to nonpulsed cells (Figure 1B). The percent specific lysis was evident even at a very low ratio of 0.625:1 Effector: Target cells (E:T). As controls, T2 cells pulsed with PR1 showed dose-dependent killing by PR1-CTLs, whereas T2 cells pulsed with CG1 and nonpulsed did not, as expected (Figure 1B).To evaluate whether NET-associated NE and P3 facilitate uptake in B-ALL, healthy PMNs were co-cultured with B-ALL cell lines overnight. The co-culture resulted in both NE (Figure 2A-B) and P3 (Figure 2C-D) uptake in RS4 and Nalm6 cell lines with an average MFI of 350 and 426, respectably, compared to nonpulsed MFI of 145. Measurement of intracellular P3 and NE by flow cytometry showed that although both NET-associated and PMN-associated NE and P3 are taken up, uptake of supernatants from resting PMNs was significantly greater than NET-associated (Figure 2). Similar to uptake of soluble and PMN-associated proteases, uptake between B-ALL cell lines was heterogeneous.ConclusionCollectively, our data demonstrate that B-ALL cell lines are capable of PR1 cross-presentation rendering them susceptible to killing by PR1-CTLs. Further, NET-associated NE and P3 are a source of protease uptake. Because NET-associated antigens are less efficient at uptake, our data suggest that NETs produced during systemic inflammation might be less immunogenic in B-cell malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Acute Lymphoblastic Leukaemia (ALL) Cells and Commonly-Used Chemotherapy Drugs Can Induce Bone Marrow-Derived Mesenchymal Stromal Cells to Become Cancer Associated Fibroblasts (CAF), Leading to Enhanced ALL Proliferation and Treatment Resistance

To investigate the role of mesenchymal stromal cells (MSCs) in treatment resistance in acute lymphoblastic leukaemia (ALL) we systematically isolated and studied, ISCT-confirmed, bone-marrow-derived MSCs isolated from 151 adults (22-69 years) with ALL registered on the UKALL14 and ALL60+ trials both at diagnosis and during therapy.CD11b, CD68 and CD163-expressing tumour-associated macrophages were observed in 34/75 (45%) of MSC cultures from follow-up samples (termed M+MSC). By contrast, macrophages were absent (M-MSC) from all diagnostic (0/56) and healthy donor specimens (0/7) tested. ELISA on supernatant from expanded M+MSC showed significantly higher levels of pro-inflammatory cytokines IL8 (4961 pg/ml vs 1093 pg/ml, p = 0.01) and CXCL2 (106 pg/ml vs 36 pg/ml, p = 0.03) than that from expanded M-MSC. The B-ALL cell line Nalm6 proliferated more rapidly when co-cultured with primary M+MSCs than with primary M-MSC (1.93 fold, p = 0.03). Importantly, primary M+MSCs strongly expressed α-smooth muscle actin (αSMA) by immunostaining, consistent with a cancer associated fibroblast (CAF) phenotype.To establish the role of ALL cells and/or chemotherapy in the induction of CAFs, we used the MSC cell line HS27a to model CAF generation in-vitro . Co-culture of HS27a with B-ALL cell lines Nalm6, SD1 and 697 cells induced increased aSMA expression and cytoskeleton remodeling including increased actin stress fibers in the HS27a consistent with a CAF phenotype. These changes were not observed in HS27a in co-culture with healthy donor B-cells or the B-ALL cell line SEM. A targeted RQPCR gene panel showed up-regulation of CAF-defining genes (FAPA 2-4.4.6 fold, COX2 5.9 - 32.1 fold, IL6 11.2 - 495 fold, FN 2.2 - 3.0 fold, TGFβ1 2.0 - 4.6 fold, IL8 15.5 - 85.2 fold, CCL2 10.6 - 33.3 fold, VEGF 2.9 - 13.4 fold, FGF2 2.9 - 14.6 fold) and expected down-regulation of CAV1 (1.8 - 5.4 fold) when HS27a were cultured with SD1 and 697 cells compared to HS27a alone. HS27a exposed to healthy donor B-cells or SEM cells in contrast did not induce differential regulation of CAF defining genes. In a flow-based, cytokine bead assay, SD1 cells induced HS27a to secrete pro-inflammatory cytokines (IL8, IL6, CCL2, CXCL10 and CCL5) and growth factor bFGF at several fold higher levels than normal B-cells or SEM cells consistent with a CAF secretion profile. Similar morphological changes, gene expression profile and cytokine profile were observed when cells were co-cultured in a transwell system, suggesting a soluble mechanism of CAF induction. TGFβ1 and reactive oxygen species, known to induce CAFs in solid tumors, were both increased in all B-ALL cell lines able to induce CAFs compared to SEM cells, suggesting a probable mechanism behind CAF induction.Therapeutically-relevant concentrations of chemotherapy agents cytarabine and daunorubicin - but not vincristine and dexamethasone - induced gross morphological changes in HS27a cells with an increase in actin stress fibres and αSMA. RQPCR of cytarabine-treated HS27a showed upregulation of several genes consistent with a CAF phenotype and cytokine bead assay showed increased secretion of IL8 and CCL2 compared to untreated HS27a (973 pg/ml vs 430pg/ml, p < 0.0001 and 1089pg/ml vs 717.3pg/ml p = 0.02 respectively).To examine the functional significance of CAF on ALL cell proliferation and chemoresistance, we pre-treated HS27a with cytarabine to induce a CAF phenotype and then assessed the impact on SEM (selected for their lack of CAF induction) proliferation and chemosensitivity. Pretreatment with vincristine or dexamethasone and untreated HS27a comprised the controls. Cytarabine-generated, HS27a-derived CAF increased SEM cell proliferation compared to both the untreated and vincristine and dexamethasone pre-treated HS27a (1.2 fold p = 0.002, 1.3 fold p = 0.0004 and 1.2 fold p = 0.0002 respectively). Cytarabine-generated, HS27a-derived CAF also reduced the sensitivity of SEM cells to both dexamethasone and cytarabine compared to both the untreated and dexamethasone or vincristine pre-treated HS27a.We have demonstrated for the first time in both primary cells and cell-line models that bone-marrow derived MSCs can adopt a CAF phenotype in response to both ALL cells and ALL therapies. The functional impact is to enhance both ALL cell proliferation and chemotherapy resistance. Ongoing work will delineate the precise nature of the interaction between MSC and ALL cells. DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Ca2+ Oscillations and NFATC1 -Activation Enable Oncogenic Signaling in Pre-B Cell Transformation and Leukemogenesis

Background: Nuclear factor of activated T cells (NFATC1) regulates activation and differentiation of B and T cells. Activation of NFATC1 requires sustained Ca2+ influx by store-operated Ca2+ entry (SOCE). Our group previously showed that oncogenic signaling from the BCR-ABL1 kinase in Ph+ ALL results in autonomous oscillatory Ca2+ signaling (Klein et al., J Exp Med 2004; Feldhahn et al., J Exp Med 2005). However, the significance of Ca2+ oscillations remained unclear. Here we examined the function of Orai and Stim1/2, the central mediators of SOCE, and the role of Ca2+ oscillations in the survival of BCR-ABL1 -driven pre-B ALL cells.Results: To investigate the consequence of inducible deletion of SOCE-effector genes, Orai1, Stim1, and Stim2, we have generated BCR-ABL1 - and CreERT2-transduced murine pre-B Orai1f/f and Stim1/2f/f cells. Both Orai1 - and Stim1/2 -deletion impaired Ca2+ influx from the extracellular space when Ca2+ uptake from cytosol to endoplasmic reticulum was pharmacologically inhibited. Consistently, both Orai1 - and Stim1/2 -deletion significantly attenuated the amplitude and frequency of autonomous Ca2+ oscillations, compared with that of empty vector controls. To determine whether the impaired Ca2+ signaling associated with cell survival and proliferation, we next analyzed their growth and colony-forming capacity. As expected, both Orai1 - and Stim1/2 -deleted pre-B ALL cells exhibited growth disadvantage and reduced colony-forming capacity, compared with empty vector controls. Flow cytometry studies of Annexin V revealed that both Orai1 - and Stim1/2 -deletion induced apoptosis in the BCR-ABL1 -transformed cells. These results suggest that autonomous Ca2+ oscillations are needed to circumvent apoptosis, and enhance cell growth and self-renewal in BCR-ABL-driven ALL cells. Interestingly, even in normal pre-B cells and in the absence of autonomous Ca2+ oscillations, both Orai1 - and Stim1/2 -deletion caused growth disadvantage suggesting the existence of similar cellular function to Ca2+ oscillations in normal pre-B cells. Subsequently, we studied NFATc1 activity by Western blot, as reflected by nuclear localization, in pre-B ALL cells before and after Orai1 - and Stim1/2 -deletion. NFATc1 is known as one of a central NFAT family member that is activated in lymphocytes upon BCR and TCR stimulation. While Orai1fl/fl and Stim1/2fl/fl B-lineage ALL cells transduced with empty vector controls displayed marked nuclear accumulation of NFATc1, Cre-mediated deletion of Orai1 and Stim1/2 strongly reduced nuclear accumulation of NFATc1, with slight increases in the cytoplasmic fraction. To examine the consequences of NFATc1 inhibition, we generated BCR-ABL1 - and CreERT2-transduced murine pre-B Nfatc1f/f and observed its effect on cell growth. As expected, inducible ablation of Nfatc1 resulted in rapid depletion of pre-B ALL cells from the cell culture compared to empty vector controls. Importantly, a NFAT-calcineurin association inhibitor, INCA-6, displayed growth inhibitory effects in all six patient-derived xenograft of B cell ALL tested, including samples from patients who relapsed after initially successful chemotherapy. These results suggest that the Ca2+-NFAT signal may suppress apoptosis, and promote drug-resistance and relapse in human pre-ALL cells.Conclusions: Taken together, our findings showed that SOCE has a critical role in NFAT activation and survival of BCR-ABL1 -driven pre-B ALL cells. Orai1- and Stim1/2-mediated autonomous Ca2+ oscillatory signal is required for BCR-ABL1-dependent proliferation. This signal may bypass the NFAT activation which suppress apoptosis and enhance proliferation in BCR-ABL-driven ALL cells. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometry Immunophemotyping Evaluation in the Diagnosis of Acute Lymphoblastic Leukemia in Patients from Rio Grande Do Norte State, Brazil

The authors conducted a flow cytometry immunophenotyping study in patients with acute lymphoblastic leukemia (ALL) from Natal, Rio Grande do Norte state, Brazil. The patients (n=126) were newly diagnosed using a panel of monoclonal antibodies: CD1a, CD2, CD3, CD4, CD7, CD8, CD10, CD13, CD33, CD14, CD19, CD22, CD79a, CD117, CD34, anti-IgM, anti-TdT, anti-HLA-Dr, anti-human kappa and lambda light chains. Additional data, such as patient age and gender, clinical and laboratory findings such as the presence of tumor masses, lymphadenopathy, hepatomegaly, splenomegaly, leukemic infiltration into the central nervous system (CNS) were also investigated. Results showed that 56.7% of the cases were B-lineage ALL and 55% T-cell ALL. Moreover, we found that males were more affected by the disease, regardless of immunological classification. The correlation between age and immunological subtypes showed that the B-lineage ALL occurred more frequently in patients younger than 15 years of age, while the T-cell ALL subtype was more frequent in adults. Immunophenotypic profiles and morphological subtypes demonstrated a direct correlation between L3 subtype and B-lineage ALL, while L1 and L2 subtypes correlated more often with B-cell lineage and T-cell ALL, respectively. Correlation analysis between immunophenotypic and clinical profiles revealed that T-cell ALL was more associated with a higher incidence of lymphadenopathy, hepatomegaly, splenomegaly and CNS leukemic infiltration, in addition to showing a greater blast cell count in peripheral blood than the other subgroups. The data suggest that immunophenotyping is an important method in diagnosis, monitoring and prognosis when determining the pathological mechanisms of evolution in acute lymphoblastic leukemia. DisclosuresNo relevant conflicts of interest to declare.

Defining the Cell Surface Proteome of MLL Rearranged Leukemia

Introduction: B-acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr ) have a particularly dismal clinical outcome. Although it is known that this oncoprotein radically alters epigenetic regulation, the precise molecular mechanisms that define the cellular phenotype of these cancers remain poorly understood and new therapeutic strategies are sorely needed. Furthermore, although flow cytometric immunophenotyping has been performed for decades on B-ALL blasts, relatively little is known about the cell surface proteome of these cancers outside of canonical CD markers. Due to its role in modulating cellular behavior as well as accessibility to immunotherapy, the cell surface represents an attractive target for unbiased proteomic profiling in order to gain insight into cellular behavior as well as enabling the development of targeted therapies. Here, we aim to resolve the cell surface proteomic landscapes of MLLr and non- MLLr B-ALL cellular models. Our goal is to generate new mechanistic insights into the molecular re-wiring that occurs in these forms of cancer and identify novel candidates for targeted immunotherapy.Methods: We examined 10 B-ALL cell lines that harbor different oncogenic fusions including the MLL-AF4, BCR-ABL, and ETV6-RUNX1 translocations, respectively, using both “classical” lines as well as low-passage patient-derived lines, in addition to non-malignant B-cells as a control. We performed unbiased labeling of cell surface proteins by biotinylation of glycosylated amino acids (3 biological replicates each) followed by affinity pull-down with streptavidin. After on-bead trypsin digestion, peptides from bound glycoproteins were analyzed on a Thermo Q-Exactive Plus Mass spectrometer using 4-hour LC runs, data-dependent acquisition, and label-free quantification to identify and quantify members of the cell surfaceome for each cell line. To confirm our proteomic findings we performed flow cytometry using a BD CytoFLEX flow cytometer on select cell lines with fluorescently conjugated monoclonal antibodies, in order to assess presence and qualitative abundance of particular cell surface targets.Results: We quantified on average >900 high-confidence membrane proteins (FDR=0.05) per cell type, comprising ~40% of all proteins identified in each experiments. Principal component analysis identified unique cell surfaceome signatures that distinguish several of the leukemia subtypes and normal B-cells from each other, implying different cell-surface phenotypes associated with specific B-ALL genetic alterations (Figure 1A). As positive controls, we found several proteins differentially expressed in MLLr that were also previously identified based on transcriptomic profiling of MLLr patient samples, such as FLT3 and PROM1. In our data, the MLLr surfaceome was surprisingly enriched for Ephrin receptors, matrix metalloproteases, and other proteins that play a role in cell motility, invasion of tissues, and modulation of the tumor microenvironment (significantly enriched membrane proteins colored in red, Figure 1B). Using flow cytometry with monoclonal antibodies specific for select cell surface markers we found excellent agreement with our proteomic data. We will next move to patient-derived xenografts and tissues for further validation. These findings suggest these novel markers may be candidates for downstream immunotherapy development, potentially specific to MLLr B-ALL, where there is particularly high clinical need.Summary and Conclusions: In this work, we profiled the cell surface proteomes of multiple B-ALL cell models, identifying cell surfaceome signatures unique to different B-ALL subtypes. Overall, our work demonstrates the power of performing un-biased interrogation of the tumor surfaceome to identify new therapeutic targets for immunotherapy, as well as provide a resource to advance our phenotypic understanding of these aggressive cancer subtypes. [Display omitted] DisclosuresWiita:TeneoBio, Inc.: Research Funding; Sutro Biopharma: Research Funding.

Genomic and Epigenetic Effects of DNA Methyltransferase Inhibition in Acute Lymphoblastic Leukemia

Background: Children with acute lymphoblastic leukemia (ALL) who relapse are more resistant to chemotherapy. We have previously identified a unique gene expression signature associated with relapsed leukemic blasts and showed that relapse samples have higher level of CpG methylation compared to diagnosis. We also demonstrated that treatment with DNMT inhibitor, decitabine, alone or in combination with the HDAC inhibitor, vorinostat restored blast chemosensitivity in vitro . These observations led to the hypothesis that global methylation status is a key regulator of drug resistance.Method: To discover the pathways affected by changes in global methylation and chromosomal architecture, we performed ChIPseq for the CCCTC-binding factor (CTCF) and the histone marks H3K9ac, H3K27ac and H3K27me3 as well as RNAseq, and DNA methylation in the B-ALL cell line Reh treated with 1µM decitabine versus DMSO for 3 days in triplicates. ChIPseq data was analyzed using MACS2 broad setting for peak calling, GENCODE for gene annotations and diffbind for differences between the two groups. Promoter regions were inferred to be +/-3KB from TSS and the ROSE algorithm was used on the H3K27ac mark to define enhancers and superenhancers. Analysis of differential gene expression was performed using DESeq2 Bioconductor package and the 850k methylation data was analyzed using ‘Minfi’ Bioconductor package. For all data sets, fold changes of 1.5 or more and p<0.05 (FDR of 5% for the ChIPseq data) were considered significant.Results: As expected, decitabine treatment resulted in a global loss of CpG methylation and there were 1286 genes upregulated and 1003 genes downregulated by 1.5 fold or more in treated versus untreated cells. Interestingly, there was a subset of CpG sites which had a significant gain of methylation, and gene ontology biological process analysis of those sites using DAVID Bioinformatics identified, among others, genes involved in mitotic cell cycle and MAPK activation. ChIPseq data analysis show that more than 90% of the peaks remain unchanged for the activation mark H3K27ac and the repressive mark H3K27me3 between decitabine-versus DMSO-treated Reh cells. However decitabine treatment resulted in a slight variation in the genomic distribution of the overall peaks. H3K27ac mark occupied 28% of the promoter region after decitabine treatment as compared to 23% in untreated state and likewise H3K27me3 occupancy changed from 11% to 8% at the promoter region with treatment with decitabine. The most impacted histone mark was H3K9ac where 40% of the global peaks called in the untreated cells were lost upon DNMTi treatment. However most of the losses of the H3K9ac mark were located outside of promoters resulting in 56% of these activation marks in promoter regions in treated cells compared to 35% in the untreated cells. Unlike the expected gain of CTCF from lower DNA methylation, there was a 30% reduction of CTCF binding sites with decitabine treatment. CTCF was disproportionately lost at promoter regions compared to gene body and intergenic regions. Integrative analysis of changes in gene expression, CpG methylation, histone marks reveal that up to 60% of their gene expression changes can be directly attributed to the observed epigenetic changes with the greatest impact from changes in DNA methylation and H3K9ac. The gene expression data was also compared to our previously published relapsed gene expression signature on a large cohort of diagnosis-relapse primary samples. We found that decitabine reversed the expression of 74 genes which are typically downregulated in blasts at relapse and 57 genes which are typically upregulated in relapsed blasts. Decitabine treatment led to two predominant epigenetic modifications: loss of CpG methylation with corresponding de-repression of genes typically downregulated in relapse samples (primarily proapototic and antiproliferative genes) and loss of H3K9ac at promoters of antiapoptotic genes.Conclusions: We show that DNMTi not only impacts CpG methylation but also histone status, likely chromosomal architecture, and gene expression in B-ALL cells and can reverse expression of a subset of the relapse gene expression signature. Further study and validation will lead to a better description of epigenetic events in relapsed ALL that may guide future therapy. DisclosuresNo relevant conflicts of interest to declare.

Obesity-Induced Chronic Inflammation Suppresses the Function of Non-Malignant Immune Cells While Upregulating PD-L1 on Human B-ALL Cell Lines

Obesity incidence continues to rise dramatically throughout the world. Currently, 20% of school-aged children (ages 6-19) and 35% of the adult population (> 20 years of age) in the United States are obese. Obesity-related complications contributes to various diseases including insulin resistance, type 2 diabetes, and cardiovascular diseases. In addition to these diseases, obesity is a risk factor for developing childhood leukemia and significantly increases mortality rates in pediatric patients. Obesity-associated chronic inflammation is postulated to contribute to the onset and propagation of leukemia; however, this hypothesis has remained largely unexplored in experimental settings. Therefore, we established an in vitro system to delineate how non-malignant immune cells and B-cell leukemia lines respond to cytokine stimulation. Specifically, dendritic cells (DC) were isolated from the spleens of C57BL/6 mice using anti-CD11c microbeads followed by MiniMACS purification and stimulated with IL-1β, IL-6, TNF-α, or LPS (as single agents or in combination) for 24 hours prior to assaying for the upregulation of CD80,CD86, and PD-L1 using flow cytometric analysis. We found that splenic DC stimulated with IL-1β or TNF-α upregulated both CD80 and CD86 in a dose-dependent manner. Surprisingly, both cytokines also induced PD-L1 expression on DC. We next tested the effects of cytokine stimulation on Nalm6, REH, and SEM human B-ALL cell lines. Only SEM cells expressed CD86, which was not modulated by cytokine stimulation. In contrast, stimulating REH and SEM cells with cytokines upregulated PD-L1 expression with TNF-α being the most potent inducer of the immune inhibitory receptor. In order to mimic a chronic inflammatory microenvironment in vitro, mouse DC cells were treated with individual pro-inflammatory cytokines for 24 hours prior to stimulation with LPS treatment for 24 hours before assaying for functional outcomes at 48 hours post-stimulation. We observed that LPS, IL-1β, and TNF-α treatments increased CD80 and CD86 expression. Surprisingly, LPS upregulation of both CD80 and CD86 was blocked on DC when cytokine treatment preceded LPS stimulation. Similarly, DC exposure to cytokines prior LPS treatment failed to induced CD40 and PD-L1 upregulation. To examine how adipocytes-produced inflammatory cytokines modulated these functional responses in non-malignant B cells, we isolated mouse B cells from spleens of C57BL/6 mice using anti-B220 microbeads followed by MiniMACS purification. Using a modified insulin Oleate (IO) media differentiation protocol, we were able to induce adipocyte differentiation from OP9 cells (a mouse bone marrow stromal cell line). Adipocytes differentiated in vitro morphologically phenocopied primary adipocytes, and they expressed a hallmark adipocyte marker Fatty Acid Binding Protein 4 (FABP4). Importantly, these cells produced copious amounts of TNF-α. When splenic B-cells were cultured with adipocyte-produced supernatants, we found that CD86 surface expression was downregulated while PD-L1 expression increased. In conclusion, we found that IL-1β and TNF-α potently induced CD80 and CD86 on splenic DC and B-cells in acute settings. Surprisingly, these cytokines also induced PD-L1 expression on these cells most likely to prevent the pathological effects of an uncontrolled immune response. In the chronic settings, we observed blunted innate immune responses to LPS stimulation. These observations suggest that chronic inflammatory microenvironments (such as those associated with obesity) will induce an immune suppressive state in splenic DC and B-cell populations. This hypothesis was supported by our observations that adipocyte-produced soluble factors also decreased CD86 expression and upregulated PD-L1 on splenic B-cells. Pro-inflammatory cytokines also potently induced PD-L1 expression on B-ALL cells. In all, our data suggest that chronic inflammation may promote leukemogenesis by both attenuating de novo immune responses and reducing the ability to effector T-cells to kill leukemia cells when engaged due to the upregulation of immune suppressive mechanisms. Disclosures No relevant conflicts of interest to declare.