Patient-derived Acute Lymphoblastic Leukemia Research Articles

BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

Targeting WEE1 kinase as a p53-independent therapeutic strategy in high-risk and relapsed acute lymphoblastic leukemia

BackgroundOutcomes for patients with relapsed acute lymphoblastic leukemia (ALL) are poor and there is a need for novel therapies to improve outcomes. Targeted inhibition of WEE1 with small-molecule inhibitor adavosertib (AZD1775) has emerged as a therapeutic strategy to sensitize cancer cells to DNA-damaging chemotherapeutics, particularly in the context of TP53-mutated tumors. However, WEE1 inhibition as a potential therapeutic strategy for patients with high-risk and relapsed ALL, including those with TP53 mutations, has not been definitively evaluated.MethodsAnti-leukemic effects of adavosertib were investigated using a relapsed TP53 isogenic cell model system, primary patient, and patient-derived ALL samples (n = 27) in an ex vivo co-culture model system with bone marrow-derived mesenchymal stem cells. Combination effects with drugs currently used for relapsed ALL were quantified by Excess over Bliss analyses. Investigations for alterations of cell cycle and apoptosis as well as related proteins were examined by flow cytometry and Western blot, respectively.ResultsOur study demonstrates the potent anti-leukemic activity of the clinically advanced WEE1 inhibitor adavosertib in a large majority (n = 18/27) of high-risk and relapsed ALL specimens at lower than clinically attainable concentrations, independent of TP53 mutation status. We show that treatment with adavosertib results in S-phase disruption even in the absence of DNA-damaging agents and that premature mitotic entry is not a prerequisite for its anti-leukemic effects. We further demonstrate that WEE1 inhibition additively and synergistically enhances the anti-leukemic effects of multiple conventional chemotherapeutics used in the relapsed ALL treatment setting. Particularly, we demonstrate the highly synergistic and cytotoxic combination of adavosertib with the nucleoside analog cytarabine and provide mechanistic insights into the combinational activity, showing preferential engagement of apoptotic cell death over cell cycle arrest. Our findings strongly support in vivo interrogation of adavosertib with cytarabine in xenograft models of relapsed and high-risk ALL.ConclusionsTogether, our data emphasize the functional importance of WEE1 in relapsed ALL cells and show WEE1 as a promising p53-independent therapeutic target for the improved treatment of high-risk and relapsed ALL.

Targeted inhibition of protein synthesis renders cancer cells vulnerable to apoptosis by unfolded protein response

Cellular stress responses including the unfolded protein response (UPR) decide over the fate of an individual cell to ensure survival of the entire organism. During physiologic UPR counter-regulation, protective proteins are upregulated to prevent cell death. A similar strategy induces resistance to UPR in cancer. Therefore, we hypothesized that blocking protein synthesis following induction of UPR substantially enhances drug-induced apoptosis of malignant cells. In line, upregulation of the chaperone BiP was prevented by simultaneous arrest of protein synthesis in B cell malignancies. Cytotoxicity by immunotoxins—approved inhibitors of protein synthesis—was synergistically enhanced in combination with UPR-inducers in seven distinct hematologic and three solid tumor entities in vitro. Synergistic cell death depended on mitochondrial outer membrane permeabilization via BAK/BAX, which correlated with synergistic, IRE1α-dependent reduction of BID, accompanied by an additive fall of MCL-1. The strong synergy was reproduced in vivo against xenograft mouse models of mantle cell lymphoma, Burkitt’s lymphoma, and patient-derived acute lymphoblastic leukemia. In contrast, synergy was absent in blood cells of healthy donors suggesting a tumor-specific vulnerability. Together, these data support clinical evaluation of blocking stress response counter-regulation using inhibitors of protein synthesis as a novel therapeutic strategy.

Block of Counter-Regulation By Inhibition of Protein Synthesis Sensitizes Cancer Cells to IRE1α-Mediated Apoptosis Following Unfolded Protein Response

Background: Cellular stress responses like the unfolded protein response (UPR) decide over the fate of an individual cell. To prevent cell death, UPR induces expression of protective proteins that aim to restore homeostasis. A similar strategy is used by cancer cells to induce resistance against UPR-inducing drugs. Therefore, we hypothesized that a simultaneous block of protein synthesis prevents UPR counter-regulation and thus, enhances UPR-induced apoptosis. Methods: Tumor cell lines and patient-derived acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells were treated with several protein synthesis inhibitors and UPR-inducers. Synergy was determined as fold-increase over additivity. Mechanistic studies were performed using western blots, shRNA-mediated knock-down, and CRISPR/Cas9-mediated knock-out. In vivo synergy was tested using intravenously injected xenografts. Results: 2-deoxyglucose (2-DG), which competitively inhibits protein glycosylation in the endoplasmic reticulum (ER), induced UPR in B cell lymphoma cell lines. An additional block of protein synthesis by CD22-targeted immunotoxin Moxetumomab pasudotox (Moxe) prevented the upregulation of the protective binding immunoglobulin protein (BiP), although UPR signaling remained active. In line, 2-DG synergistically enhanced activity of protein synthesis inhibitors Moxe, cycloheximide, and puromycin suggesting a conserved mechanism of action. Furthermore, drug synergy was not only reversed by addition of mannose, which restores proper glycosylation, but was also mimicked by the two other UPR-inducers tunicamycin and bortezomib indicating UPR as cause of synergy. Mechanistically, Moxe and 2-DG induced synergistic mitochondrial apoptosis, which correlated with synergistic cleavage of PARP, synergistic reduction of BID, and additive fall in MCL-1. Knock-down of several UPR-associated proteins identified Inositol-requiring enzyme 1α (IRE1α) as responsible protein for synergy. Knock-out of IRE1α reversed synergistic cell death, PARP cleavage, and reduction of BID but not the fall of MCL-1 confirming IRE1α as key mediator of synergy. Suggesting broad applicability, immunotoxins and 2-DG induced synergy in seven distinct hematologic and three solid tumor entities, while healthy blood cells were insensitive towards the combination. Because UPR-induced cell death depends on a persisting stress signal and 2-DG has a short half-life, exposure time in vivo was increased by four injections of 2-DG per day. Against a systemic mantle cell lymphoma model, only high frequent 2-DG treatment, but not daily single injections, enhanced Moxe activity by 3-fold. The in vivo synergy was reproduced by combination with tunicamycin and bortezomib. Furthermore, the combination of 2-DG and Moxe induced a 35-fold synergy against a systemic Burkitt lymphoma model and an up to 37-fold synergy against two patient-derived B-ALL xenografts of the Burkitt's type. Conclusion: Blocking UPR counter-regulation by simultaneous inhibition of protein synthesis induces synergistic cell death in several malignancies. Synergy depends on IRE1α-mediated deregulation of BID which enhances MCL-1-mediated mitochondrial apoptosis. The broad applicability suggests a cancer cell-specific vulnerability which, together with a cell-targeted arrest of protein synthesis by immunotoxins, generates a unique therapeutic window supporting clinical evaluation.

Targeting TPC2 sensitizes acute lymphoblastic leukemia cells to chemotherapeutics by impairing lysosomal function

Despite novel therapy regimens and extensive research, chemoresistance remains a challenge in leukemia treatment. Of note, recent studies revealed lysosomes as regulators of cell death and chemotherapy response, suggesting this organelle is a novel target for chemosensitization. Interestingly, drug-resistant VCR-R CEM acute lymphoblastic leukemia (ALL) cells have an increased expression of the lysosomal cation channel Two-Pore-Channel 2 (TPC2) compared to drug-naïve CCRF-CEM ALL cells. Concurrently, knockout (KO) of TPC2 sensitized drug-resistant VCR-R CEM cells to treatment with cytostatics. The chemosensitizing effect could be confirmed in several cell lines as well as in heterogeneous, patient-derived xenograft ALL cells, using the pharmacological TPC2 inhibitors naringenin and tetrandrine. We reveal that a dual mechanism of action mediates chemo sensitization by loss of lysosomal TPC2 function. First, because of increased lysosomal pH, lysosomal drug sequestration is impaired, leading to an increased nuclear accumulation of doxorubicin and hence increased DNA damage. Second, lysosomes of TPC2 KO cells are more prone to lysosomal damage as a result of morphological changes and dysregulation of proteins influencing lysosomal stability. This leads to induction of lysosomal cell death (LCD), evident by increased cathepsin B levels in the cytosol, truncation of pro-apoptotic Bid, as well as the reversibility of cell death by co-treatment with the cathepsin B inhibitor CA-074Me in TPC2 KO cells. In summary, this study establishes TPC2 as a novel, promising, druggable target for combination therapy approaches in ALL to overcome chemoresistance, which could be exploited in the clinic in the future. Additionally, it unravels LCD signaling as an important death-inducing component upon loss of TPC2 function.

The Effect of Ursodeoxycholic acid and N-acetyl cysteine on Lymphoblast Viability

Aim: To investigate invitro ursodeoxycholic acid (UDCA) and N-acetyl cysteine (NAC) effect on blast cell viability in children newly diagnosed with acute lymphoblastic leukemia (ALL).
 Patients and Methods: Samples were obtained from 52 newly diagnosed ALL patients aged 1 to 17 years. UDCA and NAC were added at clinically relevant concentrations (0-300 micrograms) onto 5x10^5 cells treated at room temperature in a dark place. Untreated and treated cells were stained with 7-amino-actinomycin D (7AAD PE) and analyzed by flow cytometry
 Results: Median (interquartile range; IQR) blast percentage and incubation time were 90% (11) and 18 (1.5) hours, respectively. The dead/live blast cells ratio (7AAD+) was lower in lymphoblasts treated with all NAC concentrations than untreated controls (P < 0.001). The use of NAC was noted to, regardless of concentration, contribute to lymphoblasts viability. On the contrary, the dead/live blast cells ratio in samples treated with UDCA at the abovementioned concentrations was relatively high, suggesting the protective role for both hepatotoxicities and against leukemia. However, the difference was not statistically significant (P >0.05). There was also no correlation between different doses of UCDA and NAC regarding blast cell viability (P > 0.232).
 Conclusion: The present study showed that in vitro NAC use had a protective effect on lymphoblast viability in newly diagnosed ALL patients before starting chemotherapy. Patient-derived ALL cells can be successfully analyzed ex vivo in a short and different period without loss of blasts.

Preclinical Investigation of the p53-MDM2 Antagonist Idasanutlin (RG7388) Demonstrates Significant Activity in High Risk Adult Acute Lymphoblastic Leukemia

Outcomes for adult patients with acute lymphoblastic leukemia (ALL) are dismal and have not kept pace with their pediatric counterparts, with five-year survival rates of less than 45%. TP53 mutations are infrequent in ALL, but activity of the oncoprotein MDM2 may otherwise phenotypically disrupt and circumvent normal p53 function, positing the p53-MDM2 signaling axis as a potential therapeutic target for the engagement of intrinsic cell death programs. Given the clinical safety and responses to p53-MDM2 antagonist idasanutlin (RG7388) in other hematological cancers and solid tumors, we aimed to evaluate the therapeutic potential of idasanutlin in ALL. Single-agent activity of idasanutlin was investigated in 17 high-risk ALL patient and patient-derived xenograft samples (aged 4 to 51 years), both B-ALL (n=15) and T-ALL (n=2), including KMT2A-rearranged, TCF3-rearranged, and Philadelphia-positive ALL. An ex vivo coculture of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSC) was employed to support growth of the ALL blasts during short-term culture, complemented by a fluorescent image-based microscopy platform which identifies and discriminates the two cellular compartments using random forest machine learning algorithms based on cellular nuclear staining. Idasanutlin demonstrated sub-micromolar, dose-dependent anti-leukemic activity against 15 of 17 samples tested, with half maximal effective concentrations (EC50) in the range of 10 to 220 nM (mean EC50 = 45.1nM); the two exceptions were later determined to harbor homozygous inactivating TP53 mutations; p.Y220C and p.S241P within the p53 DNA binding domain. The idasanutlin EC50 concentrations determined are clinically achievable, well below the peak plasma concentrations reported in patients for other disease indications. Furthermore, idasanutlin concentrations below 10µM had no impact upon MSC survival. Conforming to p53-MDM2 auto-regulatory feedback mechanisms, we demonstrated that idasanutlin efficiently stabilized and activated p53 at the protein level within 6 hours when treated with their respective idasanutlin EC50, to a level greater than 4-fold increased relative to their respective vehicle-only controls (p=0.001, n=7). Further, p53-regulated transcriptional target gene products, MDM2 and p21, were increased by 5-fold and 2-fold respectively, validating engagement of the p53 pathway by idasanutlin (p=0.036 and 0.125, respectively). By contrast, idasanutlin did not elicit increased expression of either p53-regulated transcriptional target gene product in the identified TP53-mutant patient samples (n=2). On-target specificity of idasanutlin was further confirmed in a NALM6 isogenic cell line model, whereby the TP53 wildtype line was sensitive to idasanutlin (EC50 = 74nM) and effectively activated p53 signaling whereas the TP53 homozygous null line was highly resistant (EC50 = ~10µM). To determine whether the decreased cell numbers and engagement of p53 signaling observed were accompanied by cell death, the capacity of idasanutlin to induce apoptosis in the ALL samples was next investigated. Patient-derived ALL samples (n=6) were treated with vehicle or idasanutlin at their respective EC50s for 24 and 48 hours, and then analyzed by flow cytometry. There was an increase in annexin-V positive cells within 24 hours compared to the vehicle-only treated cells (mean±SD 14.3±6.6% vs 27.0±21.2% respectively (p=0.125). By 48 hours significant apoptosis was attained, with a mean±SD of 59.0±23.8% annexin-V positive cells compared to the mean of vehicle-treated cells at 29.1±11.6% (p=0.004). Cleaved poly(ADP-ribose) polymerase (PARP) levels were also increased greater than 3-fold compared to vehicle-only control cells as assessed by immunoblotting (p=0.045, n=3), corroborating these findings. These data emphasize the potential of pharmacologically targeting the p53-MDM2 axis in ALL, demonstrating potent, on-target, cytotoxic activity in a range of high-risk ALL cytogenetic subgroups. Taken together, these findings support further preclinical investigations into idasanutlin and other p53-MDM2 antagonists and potential combinations to improve the treatment of adult ALL. Disclosures Irving: F. Hoffmann-La Roche: Research Funding.

Therapeutic targeting of mutant p53 in pediatric acute lymphoblastic leukemia.

Alterations of the tumor suppressor gene TP53 are found in different cancers, in particular in carcinomas of adults. In pediatric acute lymphoblastic leukemia (ALL), TP53 mutations are infrequent but enriched at relapse. As in most cancers, mainly DNA-binding domain missense mutations are found, resulting in accumulation of mutant p53, poor therapy response, and inferior outcome. Different strategies to target mutant p53 have been developed including reactivation of p53’s wildtype function by the small molecule APR-246. We investigated TP53 mutations in cell lines and 62 B-cell precursor ALL samples and evaluated the activity of APR-246 in TP53-mutated or wildtype ALL. We identified cases with TP53 missense mutations, high (mutant) p53 expression and insensitivity to the DNA-damaging agent doxorubicin. In TP53-mutated ALL, APR-246 induced apoptosis showing strong anti-leukemia activity. APR-246 restored mutant p53 to its wildtype conformation, leading to pathway activation with induction of transcriptional targets and re-sensitization to genotoxic therapy in vitro and in vivo. In addition, induction of oxidative stress contributed to APR-246-mediated cell death. In a preclinical model of patient-derived TP53-mutant ALL, APR-246 reduced leukemia burden and synergized strongly with the genotoxic agent doxorubicin, leading to superior leukemia-free survival in vivo. Thus, targeting mutant p53 by APR-246, restoring its tumor suppressive function, seems to be an effective therapeutic strategy for this high-risk group of TP53-mutant ALL.

Adoptive Transfers of Plasmacytoid Dendritic Cells after Hematopoietic Stem Cell Transplantation Decrease the Risk Acute Lymphoblastic Leukemia Relapse without Increasing the Risk of Graft-Versus-Host-Disease

Despite advances in chemotherapy and hematopoietic stem cell transplantation (HSCT), the outcome of children with relapsed acute lymphoblastic leukemia (ALL) has not significantly improved over the last 2 decades. About 50% of children with relapsed leukemia still die from their disease and ALL is still the first cause of death by cancer in children. A new hope of cure for patients with chemo-resistant cancers has emerged with the development of cancer immunotherapy. However, the major risk of post-transplant immunotherapy is the exacerbation of life-threatening Graft-versus-Host Disease (GvHD) mediated by donor-derived T cells. We therefore explored the avenue of innate immune stimulation. Several reports have demonstrated that activated Natural Killer (NK) cells can control acute myeloid leukemia (AML) in transplanted patients, whereas ALL is deemed to be resistant to NK cell killing. We recently challenged this paradigm and demonstrated that the stimulation of NK cells with third-party activated plasmacytoid dendritic cells (pDC) killed most ALL cell lines and patient-derived ALL blasts. We further demonstrated the efficacy of pDC adoptive transfers to cure ALL in a humanized mouse model of HSCT. Collectively, these results uncovered for the first time the unique therapeutic potential of activated pDC as immunotherapeutic tools to stimulate NK cell anti-leukemic activity early after HSCT. The next step toward the clinical translation of pDC-based post-transplant immunotherapy is to verify that adoptive transfers of pDC do not stimulate T cells nor exacerbate GvHD in the presence of mature T cells.We designed a GMP-compliant method for in vitro expansion and differentiation of cord blood progenitors giving rise to sufficient numbers of pDC for adoptive transfers in patients. We showed that after Toll-like receptor (TLR) stimulation, these in vitro differentiated pDC displayed a phenotype of interferon producing cells (CD80neg PDL-1+) but not of antigen presenting cells (CD80+PDL-1neg). Accordingly, in vitro mixed lymphocyte reactions with purified allogeneic T cells demonstrated that TLR-activated pDC induced very low allogeneic T cell proliferation as compared with bona fide antigen presenting cells such as myeloid dendritic cells (mDC - CD11c+) or monocyte-derived dendritic cells (mo-DC) (Figure 1A). To test whether activated pDC could exacerbate GvHD in the presence of mature T cells, we used a xenoGvHD model in which human peripheral blood mononuclear cells (PBMC) were injected in immune-deficient mice (Nod/Scid/gRc-/-, NSG). We monitored GvHD 3-times a week according to a GvHD-assessment scale as previously described. Overt GvHD was characterized by cutaneous and intestinal lesions, weight loss and high numbers of human CD3+ cells in peripheral blood. Mice were sacrificed when endpoints were reached and GvHD was confirmed by immunohistochemistry and flow cytometry. Five weekly injections of TLR-activated in vitro differentiated pDC did not accelerate the GvHD onset and the severity of the lesions were not increased. We did not either observe any difference in survival between control and pDC-treated groups (Figure 1B).Collectively, our results indicate that TLR-activated pDC do not stimulate allogeneic T cells and do not increase the risk of acute GvHD in a mouse model of xenoGvHD. We therefore expect this novel pDC-based immunotherapy to be safe for transplanted patients. These data open the way for the next step: a Phase I clinical trial of in vitro differentiated pDC after transplantation for leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterizing Deformability of Drug Resistant Patient-Derived Acute Lymphoblastic Leukemia (ALL) Cells Using Acoustic Tweezers

The role of cell mechanics in cancer cells is a novel research area that has resulted in the identification of new mechanisms of therapy resistance. Single beam acoustic (SBA) tweezers are a promising technology for the quantification of the mechanical phenotype of cells. Our previous study showed that SBA tweezers can be used to quantify the deformability of adherent breast cancer cell lines. The physical properties of patient-derived (primary) pre-B acute lymphoblastic leukemia (ALL) cells involved in chemotherapeutic resistance have not been widely investigated. Here, we demonstrate the feasibility of analyzing primary pre-B ALL cells from four cases using SBA tweezers. ALL cells showed increased deformability with increasing acoustic pressure of the SBA tweezers. Moreover, ALL cells that are resistant to chemotherapeutic drugs were more deformable than were untreated ALL cells. We demonstrated that SBA tweezers can quantify the deformability of nonadherent leukemia cells and discriminate this mechanical phenotype in chemotherapy-resistant leukemia cells in a contact- and label-free manner.

Combined inhibition of receptor tyrosine and p21-activated kinases as a therapeutic strategy in childhood ALL

AbstractReceptor tyrosine kinase (RTK)-dependent signaling has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) of childhood. However, the RTK-dependent signaling state and its interpretation with regard to biological behavior are often elusive. To decipher signaling circuits that link RTK activity with biological output in vivo, we established patient-derived xenograft ALL (PDX-ALL) models with dependencies on fms-like tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor β (PDGFRB), which were interrogated by phosphoproteomics using iTRAQ mass spectrometry. Signaling circuits were determined by receptor type and cellular context with few generic features, among which we identified group I p21-activated kinases (PAKs) as potential therapeutic targets. Growth factor stimulation markedly increased catalytic activities of PAK1 and PAK2. RNA interference (RNAi)-mediated or pharmacological inhibition of PAKs using allosteric or adenosine triphosphate (ATP)-competitive compounds attenuated cell growth and increased apoptosis in vitro. Notably, PAK1- or PAK2-directed RNAi enhanced the antiproliferative effects of the type III RTK and protein kinase C inhibitor midostaurin. Treatment of FLT3- or PDGFRB-dependent ALLs with ATP-competitive PAK inhibitors markedly decreased catalytic activities of both PAK isoforms. In FLT3-driven ALL, this effect was augmented by coadministration of midostaurin resulting in synergistic effects on growth inhibition and apoptosis. Finally, combined treatment of FLT3D835H PDX-ALL with the ATP-competitive group I PAK inhibitor FRAX486 and midostaurin in vivo significantly prolonged leukemia progression-free survival compared with midostaurin monotherapy or control. Our study establishes PAKs as potential downstream targets in RTK-dependent ALL of childhood, the inhibition of which might help prevent the selection or acquisition of resistance mutations toward tyrosine kinase inhibitors.

Abstract 93: Transcriptional control of glucocorticoid responses in leukemia

Abstract Glucocorticoids (GCs) are central to all major therapy regimens for pre-B cell-derived acute lymphoblastic leukemia (ALL), but have no activity in myeloid leukemia. Such divergent responses represent an empirically established clinical standard; however, neither the mechanism by which GCs induce cell death nor the biological basis for the distinct responses in B-cell and myeloid leukemias is clear. Studying patient-derived samples revealed that NR3C1 (glucocorticoid receptor) levels were 6- to 20-fold higher in pre-B ALL compared to chronic myeloid leukemia (CML). High levels of Nr3c1 were reduced upon B- to myeloid-lineage conversion, suggesting that regulation of NR3C1 expression and GC responsiveness depend on a B-cell transcriptional program. B-cell transcription factors (e.g. PAX5, IKZF1) are critical for B-cell development, yet they are genetically lesioned in more than 80% of pre-B ALL cases. Despite such high frequency, the significance of these inactivating lesions remains elusive. Combining ChIP-seq and RNA-seq analyses, we identified a novel B-cell transcriptional program for activation of NR3C1 and its transcriptional target TXNIP (a negative regulator of glucose uptake). Reconstitution of PAX5 or IKZF1 expression in haploinsufficient patient-derived pre-B ALL cells increased NR3C1 and TXNIP levels. Conversely, expression of dominant negative mutant of PAX5 or IKZF1 abolished NR3C1 expression. Loss of Nr3c1 or Txnip in murine BCR-ABL1-driven pre-B ALL cells resulted in survival advantage in competitive growth assays. Importantly, loss of Nr3c1 or Txnip significantly elevated glucose uptake, lactate production and cellular ATP levels. These findings suggest that GCs induce cell death by exacerbating glucose and energy depletion. Notably, reconstitution of PAX5 or IKZF1 rendered haploinsufficient patient-derived pre-B ALL cells more sensitive to dexamethasone (dex) treatment. In contrast, dominant-negative PAX5 or IKZF1 largely de-sensitized pre-B ALL cells expressing wildtype PAX5 or IKZF1. These findings suggest that B-cell transcription factors set the threshold for GC responsiveness in pre-B ALL. Since relapsed ALL cells often acquire GC resistance, drug-combinations may be useful to prevent GC-resistance. As expected, loss of Nr3c1 abrogated responses to GCs. Interestingly, loss of Txnip also largely rescued GC-induced cell death in pre-B ALL cells. On this basis, we tested drug interactions between GCs and TXNIP agonists, 3-O-methylglucose (3-OMG) and D-allose. Treating patient-derived GC-refractory pre-B ALL cells with 3-OMG or D-allose strongly synergized with GC-treatment. Collectively, our findings provide a mechanistic explanation for the empiric finding that GCs are effective in the treatment of B-cell but not myeloid malignancies, and identify TXNIP as a novel therapeutic target in pre-B ALL. Note: This abstract was not presented at the meeting. Citation Format: Lai N. Chan, Zhengshan Chen, Gang Xiao, Jae Woong Lee, Kadriye Nehir Cosgun, Huimin Geng, Valeria Cazzaniga, Hilde Schjerven, Ross A. Dickins, Markus Muschen. Transcriptional control of glucocorticoid responses in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 93. doi:10.1158/1538-7445.AM2017-93

Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9.

CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.

PP2A Balances Glucose Metabolism and Foxo Activation to Maintain Cellular Redox Homeostasis in Acute Lymphoblastic Leukemia

Introduction: Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase negatively regulates a diverse set of signaling pathways promoting tumor growth. While PP2A functions as tumor suppressor in multiple types of cancer, here, we demonstrate an unexpected pro-survival role of PP2A in pre-B acute lymphoblastic leukemia (ALL) cells. By calibrating PI3K-AKT-mTOR signaling strength, PP2A regulates glycolysis rate and thereby balances energy demands against anti-oxidant protection of pre-B ALL cells. In addition, PP2A reinstates activity of FOXO factors by dephosphorylation and thereby enables the anti-oxidant function of FOXO1 and FOXO3. Consistent with previous studies, we find PP2A is dispensable for the survival of myeloid leukemia cells which indicates a lineage-specific role of PP2A. Combined with genetic study and small molecule inhibitor, we verify that regulation in patient-derived xenografts and highlight PP2A as a therapeutic target in pre-B ALL.Results: Consistent with a divergent role of PP2A in pre-B ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. Consistent with these findings, mutations in PP2A subunits are extremely rare in B cell malignancies but relatively common in solid tumors and myeloid malignancies (COSMIC). We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A and the catalytic subunit C, which leads to near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of FoxO1, FoxO3a, p70S6K and S6 ribosomal protein, which indicated elevated PI3K-Akt-mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. PI3K and mTOR inhibitors also have rescue effect on PP2A deficient ALL cells in growth-competition assay. Luciferase-labeled PP2A-deleted ALL cells showed reduced cell growth and leukemia progression after being transplanted into recipient mice. However, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. This lineage-specific role of PP2A was verified by inducible CEBPα expression to reprogram B cell lineage ALL cells into myeloid cells.Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in Ph+ ALL but not in CML cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. By employing glucose flux metabolic profiling assay using [1,2-13C2]-D-glucose tracer, we found elevated glycolysis and repressed pentose phosphate pathway (PPP) flux in PP2A-deleted pre-B ALL cells. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted pre-B ALL cells, together with decreased anti-oxidant gene expression, increased DNA damage, including H2AX phosphorylation and p53 expression. The mechanistic role of ROS downstream of PP2A was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells. The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation.Conclusion: Here we revealed an unexpected role of PP2A in maintaining redox homeostasis in pre-B ALL cells. By regulating AKT-mTOR signaling, PP2A keeps the balance of glycolysis and PPP to meet the energy demands of pre-B ALL cells and avoids extreme levels of oxidative stress. We confirmed this pro-survival role of PP2A in both genetic mouse ALL model and human Ph+ ALL-patients derived leukemia cells. These findings highlight PP2A as a therapeutic target and suggest that agents like the PP2A inhibitor LB-100 may be of interest for pre-clinical development and testing. DisclosuresNo relevant conflicts of interest to declare.

Activation of necroptosis to overcome drug resistance in leukemia

The understanding of cell death mechanisms is crucial for the development and application of novel anti-cancer therapies to avoid or circumvent drug-resistance in refractory malignancies. Impairment of apoptotic cell death plays a major role in therapy resistance and relapse of acute lymphoblastic leukemia (ALL) patients. Therefore, efforts are being directed at new agents reactivating apoptosis or inducing alternative cell death pathways such as necroptosis, a regulated form of necrosis. In a recent study published in Science Translational Medicine we show that the IAP (inhibitor of apoptosis proteins) inhibitor birinapant potently induces cell death in patient-derived ALL cells in vitro and in vivo through a receptor-interacting protein kinase 1- (RIP1) dependent mechanism. To define the cell death modality induced downstream of RIP1, we used a multicolor lentiCRISPR approach that allows simultaneous knockout of multiple genes. We observed that apoptosis and necroptosis are induced simultaneously as the inhibition of both pathways is required to restore cell viability upon birinapant treatment. This induction of dual cell death makes birinapant and other IAP inhibitors interesting agents for the treatment of refractory or drug resistant malignancies.

Wide Variability in the Time Required for Immunotoxins to Kill B Lineage Acute Lymphoblastic Leukemia Cells: Implications for Trial Design.

Recombinant immunotoxins (rITs) targeting CD22 are highly active in hairy cell leukemia, but less so in acute lymphoblastic leukemia (ALL). This study aims to understand the variable activity of an rIT against ALL toward improving responses in clinical application. We determined in vitro activity of rITs by WST-8 assays and the time needed to kill ALL cell lines and patient-derived ALL blasts by flow cytometry. The findings were translated into two systemic ALL xenograft models. Differences in time needed to kill KOPN-8 cells for distinct rITs were addressed biochemically. In vitro activity (IC50) of anti-CD22 rIT varied 210-fold from 0.02 to 4.6 ng/mL. Activity also varied greatly depending on the time ALL cells were exposed to immunotoxin from < 30 minutes to > 4 days. For KOPN-8, the difference in exposure time was related to intracellular rIT processing. We showed in newly developed ALL xenograft models, where immunotoxins have a short half-life, that the needed exposure time in vitro predicted the responses in vivo By replacing bolus dose with small doses at frequent intervals or with continuous infusion, responses were substantially improved. We confirmed exposure time variability on patient-derived ALL samples and showed a correlation between exposure time needed to reach maximal cytotoxicity in vitro and their clinical response. The exposure time needed for rITs targeting CD22 to kill ALL cells varies widely. Our results suggest that ALL patients would have a better response rate to anti-CD22 immunotoxins if treated by continuous infusion rather than by bolus injections. Clin Cancer Res; 22(19); 4913-22. ©2016 AACR.

Abstract 3548: In vivo CRISPR reveals dual activation of apoptosis and necroptosis as means to eradicate drug resistant leukemia

Abstract Dysregulation of apoptosis constitutes a general mechanism for refractory acute lymphoblastic leukemia (ALL) to escape cell death during chemotherapy. We have assessed the potential of SMAC mimetic compounds (SM) to eradicate drug resistant ALL by blocking the pro-survival cellular inhibitor of apoptosis proteins (cIAPs). On the basis of a large xenograft bank of primary ALL samples, obtained by xenotransplantation in immunodeficient NSG mice, we detected exquisite sensitivity to SM in vitro in a third of the cases tested, including drug-resistant and relapsed samples. Potent in vitro activity was highly predictive of in vivo efficacy in the xenograft model. Treatment of transplanted mice with SM not only delayed leukemia progression, but also completely eliminated leukemia burden in established disease for several weeks following treatment. SM activity required the presence of RIP1, since lentiCRISPR mediated gene knockout of RIP1 in patient-derived ALL cells blocked SM-induced cell death in vivo and in vitro. In contrast, RIP1 loss had no significant impact on the response to standard antileukemic therapies, supporting a view that RIP1-dependent death pathways are not likely to be selected against by front line chemotherapy. Furthermore, using a multicolor lentiCRISPR approach for simultaneous disruption of multiple genes, we found that downstream of RIP1, the response to SM was dependent on simultaneous execution of apoptosis and necroptosis, since genetic targeting of both cell death pathways was required to block SM-induced death. Upstream of RIP1, expression levels of TNF receptor II (TNFR2) in primary ALL correlated with sensitivity to SM, and genetic disruption of TNFR2 conferred resistance to SM treatment in vivo. Thus, SM treatment circumvents drug resistance through parallel activation of RIP1-dependent apoptosis and necroptosis, providing a strong rationale for application in salvage treatment of refractory childhood ALL. Citation Format: Júlia Aguadé-Gorgorió, Scott McComb, Gunnar Cario, Martin Stanulla, Cornelia Eckert, Jean-Pierre Bourquin, Beat C. Bornhauser. In vivo CRISPR reveals dual activation of apoptosis and necroptosis as means to eradicate drug resistant leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3548.

Protein Kinase Inhibitor H89 Enhances the Activity of Pseudomonas Exotoxin A-Based Immunotoxins.

HA22 (Moxetumomab pasudotox) is a recombinant immunotoxin (RIT), composed of an anti-CD22 Fv fused to a truncated portion of Pseudomonas exotoxin A. HA22 is in clinical trials to treat patients with hairy cell leukemia and acute lymphoblastic leukemia (ALL). LMB-11 is an improved variant of HA22 with reduced immunogenicity, has a longer half-life in the blood and high activity in vitro and in a Burkitt lymphoma model in vivo Searching for RIT enhancing combination therapies, we found the protein kinase A inhibitor H89 to enhance LMB-11 and HA22 activity 5- to 10-fold on ALL cell lines and on patient-derived ALL samples. In addition, H89 increased the activity of mesothelin-targeting RITs SS1P (38-fold) and RG7787 (7-fold) against the cervical cancer cell line KB31. Unexpectedly we found that the enhancement by H89 was not because of inhibition of protein kinase A; it was partially recapitulated by inhibition of S6K1, which led to inactivation of its downstream targets rpS6 and GSK3β, resulting in a fall in MCL1 levels. H89 increased the rate of ADP-ribosylation of eukaryotic elongation factor 2, enhancing the arrest of protein synthesis and the reduction of MCL1 in synergy with the RIT. In summary, H89 increased RIT activity by enhancing the two key events: ADP-ribosylation of eEF2 and reduction of MCL1 levels. Significant enhancement was seen with both CD22- and mesothelin-targeting RITs, indicating that H89 might be a potent addition to RIT treatment of CD22-positive ALL and mesothelin-expressing solid tumors. Mol Cancer Ther; 15(5); 1053-62. ©2016 AACR.

Abstract PR11: Targeted engagement of B cell autoimmunity checkpoints to overcome drug resistance in pediatric Ph-like ALL

Abstract Background: Unlike other cell types, B cells are selected for an intermediate level of signaling strength. Critical survival and proliferation signals emanate from the B cell receptor (BCR): If B-cells fail to express a functional BCR, signaling output is too weak, resulting in “death by neglect”. If the BCR binds to ubiquitous self-antigen, BCR signals are exceedingly strong. Both attenuation below minimum (non-functional BCR; death by neglect) and hyperactivation above maximum (autoreactive BCR) thresholds of signaling strength trigger negative selection and cell death. Rationale: Unlike any other types of cancer, we recently discovered that pre-B acute lymphoblastic leukemia (ALL) cells are bound by the same rules that also govern normal B cell selection. Oncogenic tyrosine kinases in pediatric Ph-like ALL mimic active pre-BCR signaling. The Ph-like group defines the ALL subset with particularly poor clinical outcome. Current therapy approaches are largely focused on the development of more potent tyrosine kinase inhibitors (TKI) to suppress oncogenic signaling. However resistance to TKI is developed invariably. Here, we test the hypothesis that targeting hyperactivation above a maximum threshold will selectively kill Ph-like ALL cells through a mechanism that is functionally equivalent to removal of self-reactive B cells. Results: Ph-like ALL typically originates from pre-B cells that critically depend on survival signals emanating from a functional pre-BCR. Despite their pre-B cell origin, ALL cells lack expression of pre-BCR signaling chains Igα and Igβ, indicating lack of a functional pre-BCR. Reconstitution of Igα and Igβ was indeed sufficient to induce cell death in Ph-like ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph-like ALL cells in this experimental setting. These findings suggest that Ph-like ALL cells are selectively sensitive to even incremental increases of pre-BCR signaling. Consistent with this concept, patient-derived Ph-like ALL cells express high levels of inhibitory surface receptors PECAM1, CD300A and LAIR1 that recruit and activate phosphatases SHP1 and SHIP1, which terminate pre-BCR signaling. Importantly, high expression levels of these surface receptors were strong predictors of poor outcome of patients with ALL in two clinical trials, including Ph-like ALL patients (COG; P9906). Genetic studies revealed that Pecam1, Cd300a and Lair1 were critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases SHP1 and SHIP1. Genetic deletion of Lair1, Ptpn6 or Inpp5d in BCR-ABL1 transformed ALL caused cell death in vitro and in vivo through hyperactivation of pre-BCR signaling. Testing various components of proximal pre-BCR signaling, we found that an incremental increase of SYK tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive SYK was functionally equivalent to acute activation of an autoreactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in Ph-like ALL cells. Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of SHIP1, we demonstrated that pharmacological hyperactivation of pre-BCR signaling and engagement of negative B cell selection represents a promising new strategy to overcome drug-resistance in pediatric Ph-like ALL. Conclusion: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph-like ALL, and identified targeting hyperactivation of pre-BCR signaling as a potential novel class of therapeutic strategy. This abstract is also presented as Poster B06. Citation Format: Zhengshan Chen, Huimin Geng, Clifford A. Lowell, Stephen P. Hunger, Markus Müschen. Targeted engagement of B cell autoimmunity checkpoints to overcome drug resistance in pediatric Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR11.

Abstract 51: Molecular profiling of relapsed childhood cancer

Abstract Background: Despite the overall success achieved in treating childhood cancers, a major challenge is to find alternative treatment options for those children who suffer relapse. This requires more detailed understanding of the molecular pathways responsible for maintaining and promoting an aggressive drug resistant phenotype, as well as the successful targeting of these pathways. Materials and methods: We are implementing a molecular profiling approach to individual tumours, which includes Fluidigm Biomark qRT-PCR analysis (a panel of 96 marker genes), next-generation sequencing (Ion Proton custom AmpliSeq panel of 480 genes) and automated immunohistochemistry (IHC, 30 antibodies to 25 proteins/ phosphoproteins). Results: Preliminary testing of a subset of these genes has been conducted on 12 cell lines representing the major childhood cancer subtypes, as well as 8 patient-derived primary cultures including 4 inflammatory myofibroblastic tumours, 2 diffuse intrinsic pontine gliomas and 2 acute lymphoblastic leukemia (ALL). The potential for molecular profiling to assist in drug selection was subsequently tested in high throughput in vitro drug sensitivity assays using a drug library based on the profiling data. Gene expression has also been analysed in forty five cell lines from mice bearing patient-derived ALL xenografts. The individualised mouse xenografts will allow in vivo testing of the efficacy of molecularly targeted drugs. We are now undertaking a retrospective study of diagnostic frozen and formalin fixed paraffin embedded samples from 55 paediatric cancer patients who subsequently experienced relapse. Conclusion: We have implemented a screening approach to identify molecular targets in paediatric cancer patients who have failed the best current treatments available, and for whom treatment options following relapse are extremely limited. The ultimate goal is to undertake a multicentre prospective clinical trial in paediatric patients with recurrent or refractory solid tumours or leukemias for whom targeted drug therapy will be identified using molecular profiling. Citation Format: Carol Wadham, Federica Saletta, Jennifer A. Byrne, Toby Trahair, David S. Ziegler, Geoffrey McCowage, Glenn Marshall, Michelle Haber, Murray Norris. Molecular profiling of relapsed childhood cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 51.