Acute Lymphoblastic Leukemia Xenografts Research Articles

Combination p53 activation and BCL-xL/BCL-2 inhibition as a therapeutic strategy in high-risk and relapsed acute lymphoblastic leukemia

Due to the rarity of TP53 mutations in acute lymphoblastic leukemia (ALL), p53 re-activation by antagonism of the p53-MDM2 interaction represents a potential therapeutic strategy for the majority of ALL. Here, we demonstrate the potent antileukemic activity of the MDM2 antagonist idasanutlin in high-risk and relapsed ex vivo coculture models of TP53 wildtype ALL (n = 40). Insufficient clinical responses to monotherapy MDM2 inhibitors in other cancers prompted us to explore optimal drugs for combination therapy. Utilizing high-throughput combination screening of 1971 FDA-approved and clinically advanced compounds, we identified BCL-xL/BCL-2 inhibitor navitoclax as the most promising idasanutlin combination partner. The idasanutlin-navitoclax combination was synergistically lethal to prognostically-poor, primary-derived and primary patient blasts in ex vivo coculture, and reduced leukemia burden in two very high-risk ALL xenograft models at drug concentrations safely attained in patients; in fact, the navitoclax plasma concentrations were equivalent to those attained in contemporary “low-dose” navitoclax clinical trials. We demonstrate a preferential engagement of cell death over G1 cell cycle arrest, mechanistically implicating MCL-1-binding pro-apoptotic sensitizer NOXA. The proposed combination of two clinical-stage compounds independently under clinical evaluation for ALL is of high clinical relevance and warrants consideration for the treatment of patients with high-risk and relapsed ALL.

In vivo activity of the second-generation proteasome inhibitor ixazomib against pediatric T-cell acute lymphoblastic leukemia xenografts

The overall survival rate of patients with T-cell acute lymphoblastic leukemia (T-ALL) is now 90%, although patients with relapsed T-ALL face poor prognosis. The ubiquitin-proteasome system maintains normal protein homeostasis, and aberrations in this pathway are associated with T-ALL. Here we demonstrate the in vitro and in vivo activity of ixazomib, a second-generation orally available, reversible, and selective proteasome inhibitor against pediatric T-ALL cell lines and patient-derived xenografts (PDXs) grown orthotopically in immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJAusb (NSG) mice. Ixazomib was highly potent in vitro, with half-maximal inhibitory concentration (IC50) values in the low nanomolar range. As a monotherapy, ixazomib significantly extended mouse event-free survival of five out of eight T-ALL PDXs in vivo.

Lncrna Famlf-2 Promotes Leukemogenesis Via the DHX9/c-Myc Axis in Acute Lymphoblastic Leukemia

Long noncoding RNAs (lncRNAs), defined as the transcripts longer than 200 nt without protein-coding capacity, have been found to be aberrantly expressed in diverse human diseases including leukemia. Our previous study identified Familial acute myelogenous leukemia related factor ( FAMLF) in an acute myeloblastic leukemia (AML) pedigree. Two splice variants of FAMLF, FAMLF-1 and FAMLF-2 were detected. FAMLF-1 is upgraded in AML patients and associated with poor prognosis. However, the full cDNA sequence of FAMLF-2 is not verified and its role in leukemogenesis remain unclear. Herein, we aim to investigate the functional and mechanistic roles of FAMLF-2 in leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of FAMLF-2 was at higher levels in Acute Lymphoblastic Leukemia(ALL) cell lines when compared to AML cell lines. Bone marrow cells were obtained from de novo ALL patients. qRT-PCR showed FAMLF-2 was up-regulated in ALL patients (N=115) compared to healthy controls (N=72). The FAMLF-2 expression was positively correlated with the poor prognosis in ALL patients by Kaplan-Meier analysis. These data demonstrated that FAMLF-2 is overexpressed in ALL patients and is associated with poor prognosis. To verify the full cDNA sequence of FAMLF-2, 5‘ and 3‘ rapid amplification of the cDNA ends (RACE) was performed, and we identified the 2393 nt of FAMLF-2. FAMLF-2 is predicted to not have protein-coding potential. Further subcellular analysis showed FAMLF-2 is mainly localized in the nucleus of Jurkat cells by lncRNA FISH and subcellular fractionation assay. To further verify the role of FAMLF-2 in ALL leukemogenesis, a xenograft model was established in NCG mice by subcutaneously or intravenously injection of engineered Jurkat cells (knockdown group: shNC and sh FAMLF-2, overexpression group: pCDH and FAMLF-2). We found that the size and weight of tumors in FAMLF-2 knockdown group were significantly decreased compared with the empty vector in subcutaneous ALL xenograft tumor model. and the mice in the sh FAMLF-2 groups survived longer than those in the shNC groups in introvenous ALL xenograft tumor model (sh FAMLF-2 vs. shNC: 55 days vs. 43 days, P<0.01). These data suggested that downregulation of FAMLF-2 inhibits leukemia development. Although no significant difference was found in the overall survival of mice overexpressed FAMLF-2. We observed the accelerated central nervous system(CNS) infiltration of leukemia cells when FAMLF-2 was overexpressed (clinical score at 32th day from Vector vs. FAMLF-2: 2.7 scores vs. 1.2 scores, p < 0.01 ). In vitro study revealed knockdown of FAMLF-2 inhibited cell proliferation and promoted cell apoptosis in ALL cells, and vice versa. Taken together, our clinical, mouse model, and AML cell data demonstrated that FAMLF-2 promotes leukemogenesis. RNA sequencing was performed to study how FAMLF-2 promotes ALL development. Gene Set Enrichment Analysis (GSEA) of genes downregulated by FAMLF-2 showed “Myc targets” with the highest normalised enrichment score (NES) in Jurkat cells. C-Myc is a well-recognized transcription factor that involves in leukemogenesis. To investigate whether FAMLF-2 performed direct interaction with c-Myc, we performed RNA pull-down followed by mass spectrometry analysis. Instead of c-Myc, DExH-Box Helicase 9 (DHX9) has the highest abundance. The Western blot and RNA immunoprecipitation (RIP) assay were further performed to validated the direct interaction between FAMLF-2 and DHX9. DHX9 is an RNA helicase that participates in c-Myc mRNA stability. Inhibition of FAMLF-2 suppressed DHX9 and c-Myc expression. Reduced interaction between DHX9 protein and c-Myc mRNA were observed when FAMLF-2 were downregulated. Furthermore, RNA stability assays suggested that knockdown of FAMLF-2 leads to a declined half-life of c-Myc mRNA. Collectively, these results demonstrated that FAMLF-2 regulates the stability of c-Myc mRNA through direct binding of DHX9, thus involved in the ALL development. In summary, our study revealed the previous undefined cDNA sequence of FAMLF-2, and provides new insights of FAMLF-2 in leukemogenesis via the DHX9/c-Myc axis, which might act as a prospective prognostic biological marker in ALL.

Targeting nucleolin improves sensitivity to chemotherapy in acute lymphoblastic leukemia.

Most patients with acute lymphoblastic leukemia (ALL) are treated with chemotherapy as primary care. Although the treatment response is usually positive, resistance and relapse often occur via unknown mechanisms. The purpose of this study was to identify factors associated with chemotherapy resistance in ALL. Here, we present clinical and experimental evidence that overexpression of nucleolin (NCL), a multifunctional nucleolar protein, is linked to drug resistance in ALL. NCL mRNA and protein levels were compared between cell lines and patient samples using qRT-PCR and immunoblotting. NCL mRNA levels were compared between patients of different disease stages from our clinic patients' specimens and publicly available ALL patient datasets. Cells and patient-derived xenograft mouse experiments were performed to assess the effect of NCL inhibition on ALL chemotherapy effectiveness. Analysis of patient specimens, and publicly available RNA-sequencing datasets revealed a strong correlation between the abundance of NCL and disease relapse or poor survival in B-ALL. Altering NCL expression results in changes in drug sensitivity in ALL cell lines. High levels of NCL upregulated components of the ATP-binding cassette transporters via activation of the ERK pathway, resulting in a decrease in drug accumulation inside the cells. Targeting NCL with AS1411, an NCL-binding oligonucleotide aptamer, significantly increased the sensitivity of ALL cell lines and cells/patient-derived ALL xenograft mice to chemotherapeutic drugs and prolonged mouse survival. Our results highlight NCL as a prognostic marker in B-ALL and a potential therapeutic target to combat chemotherapy resistance in ALL.

Construction of CD19 targeted dual- and enhanced dual-antibodies and their efficiency in the treatment of B cell malignancy

BackgroundT cell-redirecting bispecific antibodies establish a connection between endogenous T cells and tumor cells, activating T cells function to eliminate tumor cells without ex vivo genetic alteration or manipulation. Here, we developed a novel dual-specific antibody (DuAb) and an enhanced DuAb (EDuAb) with different stimulation signal to activate T cells, and evaluated their impact on the treatment of acute lymphoblastic leukemia (ALL).MethodsThe expression plasmids of the DuAb and EDuAb containing CD80 molecule were constructed by cloning heavy chain and light chain variable fragments from anti-human CD19 (HI19a) and CD3 (HIT3a) monoclonal antibody hybridomas, respectively. The activation and the anti-tumor efficacy of human T cells mediated by DuAb and EDuAb were evaluated in vitro. B-cell ALL xenograft NSG mouse model was established to investigate the therapeutic effect in vivo.ResultsEDuAb promoted the optimal expansion of primary human T cells with low expression of inhibitory markers in vitro than DuAb did. Both DuAb and EDuAb showed a similar capability in inducing healthy donor T cells to specifically eliminate B-ALL cell lines and primary blasts from patients. The similar ability was also observed in the patient-derived T cells. In vivo study showed that both DuAb and EDuAb significantly alleviated tumor burden and extended survival of B-ALL xenograft NSG mice. The median survival of PBS, DuAb and EDuAb treatment groups were 27, 38 and 45 days, respectively. The phenotype of T cells and cytokine release in peripheral blood (PB) of B-ALL xenograft NSG mice on day 24 were analyzed as well. The results showed that the proportion of CD8+ T cells and cytokine levels, including IL-2, IFN-γ and TNF-α, were higher in the EDuAb group than that of DuAb. Moreover, both DuAb and EDuAb significantly decreased the residual leukemia cells in PB of B-ALL xenograft NSG mice.ConclusionsBoth DuAb and EDuAb showed great potential as novel treatments for B-ALL in clinical applications. However, compared to DuAb, EDuAb showed a significant advantage in promoting the proliferation and survival of T cells. Furthermore, EDuAb showed a better promising effect on eliminating tumor cells and extending survival in vivo, which provides new insights for the development of new multi-specific antibodies.

Cynaropicrin disrupts tubulin and c-Myc-related signaling and induces parthanatos-type cell death in multiple myeloma

The majority of blood malignancies is incurable and has unforeseeable remitting-relapsing paths in response to different treatments. Cynaropicrin, a natural sesquiterpene lactone from the edible parts of the artichoke plant, has gained increased attention as a chemotherapeutic agent. In this study, we investigated the effects of cynaropicrin against multiple myeloma (MM) cells in vitro and assessed its in vivo effectiveness in a xenograft tumor zebrafish model. We showed that cynaropicrin exerted potent cytotoxicity against a panel of nine MM cell lines and two leukemia cell lines with AMO1 being the most sensitive cell line (IC50 = 1.8 ± 0.3 µM). Cynaropicrin (0.8, 1.9, 3.6 µM) dose-dependently reduced c-Myc expression and transcriptional activity in AMO1 cells that was associated with significant downregulation of STAT3, AKT, and ERK1/2. Cell cycle analysis showed that cynaropicrin treatment arrested AMO1 cells in the G2M phase along with an increase in the sub-G0G1 phase after 24 h. With prolonged treatment times, cells accumulated more in the sub-G0G1 phase, implying cell death. Using confocal microscopy, we revealed that cynaropicrin disrupted the microtubule network in U2OS cells stably expressing α-tubulin-GFP. Furthermore, we revealed that cynaropicrin promoted DNA damage in AMO1 cells leading to PAR polymer production by PARP1 hyperactivation, resulting in AIF translocation from the mitochondria to the nucleus and subsequently to a novel form of cell death, parthanatos. Finally, we demonstrated that cynaropicrin (5, 10 µM) significantly reduced tumor growth in a T-cell acute lymphoblastic leukemia (T-ALL) xenograft zebrafish model. Taken together, these results demonstrate that cynaropicrin causes potent inhibition of hematopoietic tumor cells in vitro and in vivo.

In vivo activity of the dual SYK/FLT3 inhibitor TAK-659 against pediatric acute lymphoblastic leukemia xenografts.

While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains dismal. Spleen tyrosine kinase (SYK) is a prominent cytosolic nonreceptor tyrosine kinase in pediatric B-lineage ALL (B-ALL). Activating mutations or overexpression of Fms-related receptor tyrosine kinase 3 (FLT3) are associated with poor outcome in hematological malignancies. TAK-659 (mivavotinib) is a dual SYK/FLT3 reversible inhibitor, which has been clinically evaluated in several other hematological malignancies. Here, we investigate the in vivo efficacy of TAK-659 against pediatric ALL patient-derived xenografts (PDXs). SYK and FLT3mRNA expression was quantified by RNA-seq. PDX engraftment and drug responses in NSG mice were evaluated by enumerating the proportion of human CD45+ cells (%huCD45+ ) in the peripheral blood. TAK-659 was administered per oral at 60mg/kg daily for 21 days. Events were defined as %huCD45+ ≥ 25%. In addition, mice were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival and stringent objective response measures. FLT3 and SYK mRNA expression was significantly higher in B-lineage compared with T-lineage PDXs. TAK-659 was well tolerated and significantly prolonged the time to event in six out of eight PDXs tested. However, only one PDX achieved an objective response. The minimum mean %huCD45+ was significantly reduced in five out of eight PDXs in TAK-659-treated mice compared with vehicle controls. TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric ALL PDXs representative of diverse subtypes.

A Novel bispecific T-cell engager (BiTE) targeting CD22 and CD3 has both in vitro and in vivo activity and synergizes with blinatumomab in an acute lymphoblastic leukemia (ALL) tumor model

Immunotherapy has revolutionized cancer therapy. Two recently FDA-approved immunotherapies for B-cell malignancies target CD19, in the form of a Bispecific T-Cell Engager (BiTE) antibody construct or chimeric antigen receptor T (CAR-T) cells. Blinatumomab, an FDA-approved BiTE, binds to CD19 on B cells and to CD3 on T cells, mediating effector-target cell contact and T-cell activation that results in effective elimination of target B cells. Although CD19 is expressed by essentially all B-cell malignancies at clinical presentation, relapses with loss or reduction in CD19 surface expression are increasingly recognized as a cause of treatment failure. Therefore, there is a clear need to develop therapeutics for alternate targets. We have developed a novel BiTE consisting of humanized anti-CD22 and anti-CD3 single chain variable fragments. Target binding of the anti-CD22 and anti-CD3 moieties was confirmed by flow cytometry. CD22-BiTE promoted in vitro cell-mediated cytotoxicity in a dose and effector: target (E:T)-dependent fashion. Additionally, in an established acute lymphoblastic leukemia (ALL) xenograft mouse model, CD22-BiTE demonstrated tumor growth inhibition, comparable to blinatumomab. Further, the combination of blinatumomab and CD22-BiTE yielded increased efficacy in vivo when compared to the single agents. In conclusion, we report here the development of a new BiTE with cytotoxic activity against CD22+ cells which could represent an alternate or complementary therapeutic option for B-cell malignancies.

The orally bioavailable GSPT1/2 degrader SJ6986 exhibits in vivo efficacy in acute lymphoblastic leukemia

AbstractAdvancing cure rates for high-risk acute lymphoblastic leukemia (ALL) has been limited by the lack of agents that effectively kill leukemic cells, sparing normal hematopoietic tissue. Molecular glues direct the ubiquitin ligase cellular machinery to target neosubstrates for protein degradation. We developed a novel cereblon modulator, SJ6986, that exhibits potent and selective degradation of GSPT1 and GSPT2 and cytotoxic activity against childhood cancer cell lines. Here, we report in vitro and in vivo testing of the activity of this agent in a panel of ALL cell lines and xenografts. SJ6986 exhibited similar cytotoxicity to the previously described GSPT1 degrader CC-90009 in a panel of leukemia cell lines in vitro, resulting in apoptosis and perturbation of cell cycle progression. SJ6986 was more effective than CC-90009 in suppressing leukemic cell growth in vivo, partly attributable to favorable pharmacokinetic properties, and did not significantly impair differentiation of human CD34+ cells ex vivo. Genome-wide CRISPR/Cas9 screening of ALL cell lines treated with SJ6986 confirmed that components of the CRL4CRBN complex, associated adaptors, regulators, and effectors were integral in mediating the action of SJ6986. SJ6986 is a potent, selective, orally bioavailable GSPT1/2 degrader that shows broad antileukemic activity and has potential for clinical development.

In vivo activity of the dual PI3Kδ and PI3Kγ inhibitor duvelisib against pediatric acute lymphoblastic leukemia xenografts.

Acute lymphoblastic leukemia (ALL) remains one of the most common causes of cancer-related mortality in children. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases, and aberrations in the PI3K pathway are associated with several hematological malignancies, including ALL. Duvelisib (Copiktra) is an orally available, small molecule dual inhibitor of PI3Kδ and PI3Kγ, that is Food and Drug Administration (FDA) approved for the treatment of relapsed/refractory chronic lymphocytic leukemia and small lymphocytic lymphoma. Here, we report the efficacy of duvelisib against a panel of pediatric ALL patient-derived xenografts (PDXs). Thirty PDXs were selected for a single mouse trial based on PI3Kδ (PIK3CD) and PI3Kγ (PIK3CG) expression and mutational status. PDXs were grown orthotopically in NSG (NOD.Cg-Prkdcscid IL2rgtm1Wjl /SzJAusb) mice, and engraftment was evaluated by enumerating the proportion of human versus mouse CD45+ cells (%huCD45+ ) in the peripheral blood. Treatment commenced when the %huCD45+ reached greater than or equal to 1%, and events were predefined as %huCD45+ greater than or equal to 25% or leukemia-related morbidity. Duvelisib was administered per oral (50mg/kg, twice daily for 28days). Drug efficacy was assessed by event-free survival and stringent objective response measures. PI3Kδ and PI3Kγ mRNA expression was significantly higher in B-lineage than T-lineage ALL PDXs (p-values <.0001). Duvelisib was well-tolerated and reduced leukemia cells in the peripheral blood in four PDXs, but with only one objective response. There was no obvious relationship between duvelisib efficacy and PI3Kδ or PI3Kγ expression or mutation status, nor was the in vivo response to duvelisib subtype dependent. Duvelisib demonstrated limited in vivo activity against ALL PDXs.

Abstract 2053: Structural variants detected by optical genome mapping in acute lymphoblastic leukemia patient-derived xenografts models

Abstract Background: The established classification of acute lymphoblastic leukemia (ALL) does not cover all high-risk patients because of the difficulty in detecting novel or rare structural variants (SVs). Furthermore, many SVs and formed gene fusions have adverse prognostic effects on hematological malignancies. This study aims to investigate SVs in 22 patient-derived xenografts (PDX) of ALL and to assess rare SVs and chromosomal aberrations, particularly hematological malignancy genes and gene fusions in different individuals detected by optical genome mapping (OGM). Methods: Leukemia cells from 16 patients with B-cell precursor acute lymphoblastic leukemia (B-ALL) and 6 patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into immuno-deficient mice, growth curves were monitored, and leukemia cells were collected after human CD45 was more than 80%. OGM (Bionano Genomics), RNA sequencing (RNA-seq) and whole genome sequencing (WGS) techniques were used to detect SVs and gene fusions. In validation, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) tests were performed to identify specific SVs. Results: On average, each model detected 42 rare SVs with known overlapping genes in different types by OGM: 12 insertions, 25 deletions, 1 inversion, 3 duplications, 2 inter-chromosomal translocations and 1 intra-chromosomal translocation. 5 rare SVs were found in multiple models (&amp;gt;=5), and two of them were never reported in B-ALL and T-ALL. Gene fusions were detected in all ALL models by OGM, and 17% were novel fusions. At the mRNA level, gene fusions were detected only in 19 models, and 24% of the predicted gene fusions by OGM were identical to the results of RNA-seq (high confidence results including pseudogenes). These two technologies could detect different fusion partners. For example, MLLT1 was seen at the mRNA level, and ACER1 was detected at the DNA level in the same KMT2A rearrangement model. Of the 16 B-ALL models, 6 were p190 BCR-ABL1 (a fusion of BCR exon 1 and ABL1 exon 2), 1 was p210 BCR-ABL1 (a fusion of BCR exon 13 and ABL1 exon 2) and 5 of 7 had IKZF1 gene deletions. 2 p190 BCR-ABL1 models were insensitive to imatinib and 2 p190 BCR-ABL1 models as well as the model with IKZF1 exons 4-7 deletion were sensitive to imatinib when treatment was initiated early in the disease. A novel ARL15-IKZF1 fusion was identified in one of the sensitive models. Deletion of the PRKAR2B gene was another rare SV in both models sensitive to imatinib besides the IKZF1 deletion. Conclusion: OGM assays were more comprehensive, and novel rare SVs were identified in ALL. Combining the results of gene fusions between different technologies will provide more accurate predictions for ALL classifications. The combination of aberrant SVs may synergize to influence the efficacy of imatinib, which warrants further investigations. Citation Format: Yueying Wang, Mengting Qin, JinPing Liu, Xiaobo Chen, Jia Xue, Sheng Guo. Structural variants detected by optical genome mapping in acute lymphoblastic leukemia patient-derived xenografts models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2053.

Establishment of a Patient-Derived T-Cell Acute Lymphoblastic Leukemia Xenograft Model in Novel Immunodeficient NCG Mice

The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model. Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model. On the 10th day after inoculation, hCD45+ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3+ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks. Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.

Epigenetic Fine Mapping and Functional Dissection of Inherited Non-Coding Variation Impacting the Pharmacogenomics of Acute Lymphoblastic Leukemia Treatment

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer. Despite cure rates exceeding 90%, relapsed ALL is the 5th most common pediatric cancer. Although chemotherapeutic drug resistance remains the primary cause of treatment failure and relapse, it remains unclear to what extent non-coding inherited DNA sequence variants impact gene regulatory programs that contribute to differences in chemotherapeutic drug resistance and clinical outcome in childhood ALL. To address this knowledge gap we performed epigenetic fine mapping on inherited non-coding DNA sequence variants associated with ex vivo resistance to six antileukemic agents (dexamethasone, prednisolone, l-asparaginase, vincristine, 6-mercaptopurine and 6-thioguanine) in primary ALL cells from pediatric patients and inherited non-coding DNA sequence variants associated with clinical outcome (persistence of minimal residual disease and relapse) in pediatric patient cohorts using chromatin accessibility data from over 130 primary ALL cells, ALL cell lines and ALL xenografts. This analysis fine mapped over 1700 variants (sentinel variants and variants in high linkage disequilibrium; r2>0.8) to cis-regulatory elements in the ALL genome. To comprehensively dissect the functional effects of these fine mapped variants on gene regulatory activity we performed massively parallel reporter assays (MPRAs) in 10 human ALL cell lines of both B- and T-cell lineages. MPRAs utilized 23 DNA barcodes per variant allele and tested variants at bidirectional promoters using both sequence orientations. In total, 98,168 oligonucleotides each containing 175-bp of genomic sequence centered on each variant allele and a unique 10-bp barcode sequence were evaluated for gene regulatory activity. MPRA identified 558 variants with significant and concordant allele-specific activities in three or more ALL cell lines. Expression quantitative trait loci (eQTL) mapping and HiChIP three-dimensional promoter loop profiling identified candidate target genes for a subset of functional variants. We additionally evaluated the functional effects of top ranked variants using luciferase reporter assays and CRISPR/Cas9 genome editing. These variants were associated with the expression of B3GNT5, CACUL1, HERC1, KDM4C and VRK3. Notably, two of these functional variants (rs10411204 and rs1247117) impacted transcription factor (TF) consensus DNA motifs for AP-1 family TFs and PU.1. Collectively, these data suggest that many inherited non-coding variants associated with pharmacogenomic traits in childhood ALL have significant gene regulatory effects. Importantly, the identification of chemotherapeutic drug resistance-associated molecular pathways perturbed by inherited non-coding variants can improve our ability to circumvent drug resistance with novel therapeutic approaches.

Preclinical efficacy of azacitidine and venetoclax for infant KMT2A-rearranged acute lymphoblastic leukemia reveals a new therapeutic strategy

Infants with KMT2A-rearranged B-cell acute lymphoblastic leukemia (ALL) have a dismal prognosis. Survival outcomes have remained static in recent decades despite treatment intensification and novel therapies are urgently required. KMT2A-rearranged infant ALL cells are characterized by an abundance of promoter hypermethylation and exhibit high BCL-2 expression, highlighting potential for therapeutic targeting. Here, we show that hypomethylating agents exhibit in vitro additivity when combined with most conventional chemotherapeutic agents. However, in a subset of samples an antagonistic effect was seen between several agents. This was most evident when hypomethylating agents were combined with methotrexate, with upregulation of ATP-binding cassette transporters identified as a potential mechanism. Single agent treatment with azacitidine and decitabine significantly prolonged in vivo survival in KMT2A-rearranged infant ALL xenografts. Treatment of KMT2A-rearranged infant ALL cell lines with azacitidine and decitabine led to differential genome-wide DNA methylation, changes in gene expression and thermal proteome profiling revealed the target protein-binding landscape of these agents. The selective BCL-2 inhibitor, venetoclax, exhibited in vitro additivity in combination with hypomethylating or conventional chemotherapeutic agents. The addition of venetoclax to azacitidine resulted in a significant in vivo survival advantage indicating the therapeutic potential of this combination to improve outcome for infants with KMT2A-rearranged ALL.

In vivo PDX CRISPR/Cas9 screens reveal mutual therapeutic targets to overcome heterogeneous acquired chemo-resistance

Resistance towards cancer treatment represents a major clinical obstacle, preventing cure of cancer patients. To gain mechanistic insights, we developed a model for acquired resistance to chemotherapy by treating mice carrying patient derived xenografts (PDX) of acute lymphoblastic leukemia with widely-used cytotoxic drugs for 18 consecutive weeks. In two distinct PDX samples, tumors initially responded to treatment, until stable disease and eventually tumor re-growth evolved under therapy, at highly similar kinetics between replicate mice. Notably, replicate tumors developed different mutations in TP53 and individual sets of chromosomal alterations, suggesting independent parallel clonal evolution rather than selection, driven by a combination of stochastic and deterministic processes. Transcriptome and proteome showed shared dysregulations between replicate tumors providing putative targets to overcome resistance. In vivo CRISPR/Cas9 dropout screens in PDX revealed broad dependency on BCL2, BRIP1 and COPS2. Accordingly, venetoclax re-sensitized derivative tumors towards chemotherapy, despite genomic heterogeneity, demonstrating direct translatability of the approach. Hence, despite the presence of multiple resistance-associated genomic alterations, effective rescue treatment for polychemotherapy-resistant tumors can be identified using functional testing in preclinical models.

B- and T-cell acute lymphoblastic leukemias evade chemotherapy at distinct sites in the bone marrow.

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support escape from treatment. Using three-dimensional fluorescence imaging of ten primary ALL xenografts we identified sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detected B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon shortterm chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment.

Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies.

Despite approval of B-cell lymphoma (BCL)-2 inhibitor venetoclax for certain hematologic malignancies, its broader clinical benefit is curtailed by resistance. Our study aimed to determine if treatment with novel anticancer agents targeting BCL-2 and mouse double minute 2 (MDM2) could overcome venetoclax resistance in preclinical models. Venetoclax-sensitive and venetoclax-resistant acute myeloid leukemia (AML) and acute lymphoblastic leukemia cells and xenograft models were used to evaluate antitumor effects and underlying mechanisms associated with combined BCL-2 inhibitor lisaftoclax (APG-2575) and MDM2 inhibitor alrizomadlin (APG-115). The combination exhibited synergistic antiproliferative and apoptogenic activities in TP53 wild-type AML cell lines in vitro. This synergy was further exemplified by deep antitumor responses and prolonged survival in AML cell line-derived and patient-derived xenograft models. Interestingly, the combination treatment resensitized (to apoptosis) venetoclax-resistant cellular and mouse models established via chronic drug exposure or genetically engineered with clinically relevant BCL-2 gene mutations. Synergistic effects in reducing cellular viability and proliferation were also demonstrated in primary samples of patients with venetoclax-resistant AML treated with lisaftoclax and alrizomadlin ex vivo. Mechanistically, alrizomadlin likely primes cancer cells to BCL-2 inhibition-induced cellular apoptosis by downregulating expression of antiapoptotic proteins myeloid cell leukemia-1 and BCL-extra-large and upregulating pro-death BCL-2-associated X protein. Lisaftoclax in combination with alrizomadlin overcomes venetoclax resistance mediated by various mechanisms, including BCL-2 mutations. In addition, we posit further, putative molecular mechanisms. Our data rationalize clinical development of this treatment combination in patients with diseases that are insensitive or resistant to venetoclax.

Abstract 2 Development of CAR-NK Cell Therapy for Hematologic Malignancies

IntroductionNatural killer (NK) cells can kill tumor cells without priming or prior activation through their complement of activating and inhibitory surface molecules. Chimeric antigen receptor (CAR) expression by engineered NK cells can improve both specificity and potency of the NK cells’ anti-tumor efficacy. CAR-NK cell therapy may be a safer, more clinically accessible, and cost effective allogeneic cellular therapy in comparison to autologous CAR-T cell therapy, as NK cells do not cause graft versus host disease (GvHD) or cytokine release syndrome (CRS).ObjectiveWe aimed to develop an allogeneic, cryopreserved, off-the-shelf CAR-NK cell product for the treatment of hematologic malignancies.MethodsOur CD56+ NK cells are generated from cord blood-derived CD34+ cells, which undergo expansion and priming on a proprietary Notch ligand cell culture platform followed by a second culture phase of NK cell differentiation. These cells were transduced using a viral vector to express a CAR specific for an antigen expressed on the cell surface of acute lymphoblastic leukemia (ALL) cells. The CAR-NK cells were assessed for CAR expression, NK cell phenotype, and in vitro cytotoxicity both pre- and post-cryopreservation. An ALL xenograft mouse model was treated using repeat doses of cryopreserved CAR-NK cell product, with readouts of body weight, tumor growth, and survival.ResultsThe transduced CAR-NK cell product displayed viability and phenotyping comparable to the untransduced control NK cell product but possessed significantly enhanced cytotoxicity against ALL target cells in vitro. Following cryopreservation and thaw, the CAR-NK cell product retained CAR expression and maintained enhanced anti-tumor function in vitro. Cryopreserved CAR-NK cell product was safely given in up to 8 repeat doses to ALL xenograft mice and significantly inhibited tumor growth as well as increased survival compared with control NK cell product.DiscussionWe have demonstrated in this proof-of-concept study that our cord blood CD34+ cell-derived CAR-NK cell product can maintain CAR expression, with specific and enhanced potency both in vitro and in vivo, following cryopreservation and thaw. Additional preclinical studies are planned to develop CAR-NK off-the-shelf cell therapy for AML and other hematologic malignancies.

The first Japanese biobank of patient-derived pediatric acute lymphoblastic leukemia xenograft models.

A lack of practical resources in Japan has limited preclinical discovery and testing of therapies for pediatric relapsed and refractory acute lymphoblastic leukemia (ALL), which has poor outcomes. Here, we established 57 patient-derived xenografts (PDXs) in NOD.Cg-Prkdcscid ll2rgtm1Sug /ShiJic (NOG) mice and created a biobank by preserving PDX cells including three extramedullary relapsed ALL PDXs. We demonstrated that our PDX mice and PDX cells mimicked the biological features of relapsed ALL and that PDX models reproduced treatment-mediated clonal selection. Our PDX biobank is a useful scientific resource for capturing drug sensitivity features of pediatric patients with ALL, providing an essential tool for the development of targeted therapies.

Abstract 3934: KT-253, a highly potent and selective heterobifunctional MDM2 degrader for the treatment of wildtype p53 tumors with superior potency and differentiated biological activity compared to small molecule inhibitors (SMI)

Abstract Background: The murine double minute 2 (MDM2) oncoprotein is a key E3 ubiquitin ligase that degrades the tumor-suppressor p53. Reversible SMIs of the MDM2/p53 interaction have been developed to stabilize p53 and to induce apoptosis in wildtype p53 tumors. However, MDM2 SMIs induce a p53/MDM2 feedback loop, resulting in upregulation of MDM2 protein levels and p53 pathway inhibition, thus drastically limiting their biological activity and clinical application. MDM2 targeted protein degradation suppresses p53-dependent MDM2 protein feedback upregulation and is therefore expected to lead to a superior response compared to SMIs. Objectives: We discovered KT-253, a novel, highly potent heterobifunctional MDM2 degrader. We characterized the activity and molecular mechanism of KT-253 in wildtype p53 cell lines and xenograft models and demonstrated that KT-253 has superior activity compared to SMIs in these models. Methods: We characterized KT-253 in MDM2-dependent cell lines in vitro, as well as in in vivo mouse AML and ALL xenograft models. Methods included in vitro cell proliferation and apoptosis assays, gene expression profiling, and in vivo pharmacological studies. Results: KT-253 selectively depletes MDM2 protein levels with sub-nanomolar cellular potency. We demonstrated in the Acute Lymphoblastic Leukemia (ALL) cell line RS4;11 that KT-253 can overcome the p53-dependent upregulation of MDM2 protein levels as seen for reversible SMIs. In addition, even brief exposures to KT-253 in these cells could more potently stabilize p53 compared to SMIs, leading to apoptosis mediated through p53 target genes. The superior MDM2/p53 pathway inhibition and induction of apoptosis by KT-253 translates into a &amp;gt;200-fold stronger cell growth inhibition, compared to SMIs, across a panel of solid and hematological tumor cell lines. Using mouse xenograft models, we established an intermittent dosing schedule that drives anti-tumor efficacy. Importantly, a single dose of KT-253 at 1 mg/kg resulted in sustained tumor regression in the RS4;11 mouse xenograft model. In this model, KT-253 exposures correlated with induction of apoptotic p53 target genes and tumor growth inhibition. In addition, weekly administration of KT-253 significantly prolonged the survival in a disseminated Acute Myeloid Leukemia (AML) model when compared to vehicle treated animals. Conclusions: In summary, KT-253, our development candidate, has significantly improved potency relative to reversible SMIs leading to potent in vitro and in vivo efficacy that is superior to all clinically active agents. In addition, an intermittent dosing schedule of the highly potent KT-253 degrader can induce rapid apoptosis in MDM2-dependent cancer cells potentially leading to improved efficacy and safety profile. Citation Format: Yogesh Chutake, Michele Mayo, Dapeng Chen, Bradley Enerson, Patricia Cho, Jessica Filiatrault, Crystal Brown, Michael Placke, Madison Adams, Rahul Karnik, James Shaw, Yatao Shi, Dirk Walther, Alice McDonald, Frank Qi, Phillip Liu, Joseph D. Growney, Kirti Sharma, Duncan Walker, Stefanie Schalm, Juliet Williams, Matthew Weiss. KT-253, a highly potent and selective heterobifunctional MDM2 degrader for the treatment of wildtype p53 tumors with superior potency and differentiated biological activity compared to small molecule inhibitors (SMI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3934.