Disparate In Vivo Efficacy of FTY720 in Xenograft Models of Philadelphia Positive and Negative B-lineage Acute Lymphoblastic Leukemia

Abstract CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)–like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

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Selective Inhibition of Class II HDAC Isoforms 4 and 5 Provides a Promising Therapeutic Intervention for MLL-Rearranged Acute Lymphoblastic Leukemia in Infants

Blood ◽

10.1182/blood-2021-147164 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2206-2206

Author(s):

Tamara C.A.I. Verbeek ◽

Susan Arentsen-Peters ◽

Patricia Garrido Castro ◽

Sandra Pinhancos ◽

Kirsten Vrenken ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Hdac Inhibitor ◽

Lymphoblastic Leukemia ◽

Hdac Inhibitors ◽

Selective Inhibition ◽

Class Ii ◽

In Vivo Efficacy ◽

Pdx Models

Abstract MLL-rearranged acute lymphoblastic leukemia (ALL) is characterized by deregulation of the epigenome and shows susceptibility towards epigenetic perturbators such as histone deacetylase (HDAC) inhibitors. Hence, HDACs represent attractive drug targets and a variety of small molecule HDAC inhibitors have been developed and evaluated for the treatment of hematological malignancies. However, most broad-spectrum inhibitors, which simultaneously target the majority of human HDAC isoforms, often induce toxicity, especially in combination with other therapeutic agents. Therefore, selective inhibition of only one or two HDAC isoforms may represent a better alternative, provided that disease-specific dependency on specific HDACs has been identified. We examined the effects of shRNA-mediated knock-down of the class II HDACs (i.e. HDAC4, HDAC5, HDAC6, HDAC7 and HDAC9) in the MLL-rearranged ALL cell lines SEM and ALL-PO. Except for HDAC9, loss of expression (both on the mRNA and protein level) of all HDACs led to strong reductions in viable cells (0.70 to 0.19-fold; p=0.02-0.0016) in both models due to apoptosis, cell cycle arrest, or a combination thereof. Next, we evaluated the in vitro efficacy of a variety of class II HDAC-specific inhibitors on a panel of MLL-rearranged ALL (n=5) using 4-day viability MTT assays. This revealed that the selective HDAC4/5 inhibitor LMK-235 was able to recapitulate the loss-of-function phenotype of HDAC4 and HDAC5. Dose response curves showed complete growth inhibition in MLL-rearranged ALL cell lines (n=5), as well as in primary MLL-rearranged infant ALL patient samples (n=4), with IC 50 values of ~100 nM and 40-100 nM, respectively. Importantly, at these concentrations, LMK-235 hardly affected whole bone marrow samples derived from healthy individuals (n=2), for which IC 50 values were ~1 µM. To further explore the potential of class II HDAC inhibitor-based therapeutic strategies, we performed a combinatorial drug screen to identify compounds that synergize with LMK-235. For this, a compound library (comprising >200 unique agents) was screened in the absence and presence of varying concentrations of LMK235 in the MLL-rearranged cell line models SEM and ALL-PO. This, and subsequent validation experiments in additional cell line models, revealed that Venetoclax (BCL2 inhibitor), Trametinib (MEK/ERK inhibitor), Ponatinib (multi-tyrosine kinase inhibitor) and Omipalisib (a PI3K/mTOR inhibitor) strongly synergized with LMK-235. Average ZIP synergy scores ranged from 10-30, with peak ZIP scores up to 40. Importantly, synergistic effects were consistent over all concentration combinations tested. The addition of 50-100 nM LMK-235 strongly reduced IC 50 values for Omipalisib, Ponatinib and Venetoclax (0.27-fold p=0.003, 0.11-fold p=0.0005, 0.75-fold p=0.0004, respectively) in both models. In preparation to assess the in vivo efficacy of LMK-235 in patient-derived xenograft (PDX) mouse models of MLL-rearranged infant ALL, pharmacokinetic/pharmacodynamic (PK/PD) analysis was performed in immunodeficient NSG mice (n=5). For this, mice were treated with 20 mg/kg of LMK-235, daily administered via intraperitoneal injections for a total of 29 days. While none of the mice showed signs of toxicity or weight loss, LMK-235 plasma levels were stably maintained at concentrations that are highly effective against MLL-rearranged ALL cells in vitro. Taken together, these data demonstrate that various class II HDAC isoforms are targetable vulnerabilities in MLL-rearranged ALL and that pharmaceutical inhibition of HDAC4/5 by LMK-235 represents an attractive therapeutic option. Moreover, high levels of synergy observed between this HDAC inhibitor and various agents belonging to drug classes already reported to be effective against MLL-rearranged ALL, warrants pre-clinical evaluation in vivo. Currently, the assessment of the in vivo efficacy of LMK-235 monotherapy in MLL-rearranged infant ALL PDX models is in progress, after which promising synergistic HDAC inhibitor-based drug combinations will be evaluated. To determine the additional therapeutic value, the efficacy of LMK-235 and promising synergistic combinations will be evaluated in the background of conventional combination chemotherapy, where PDX models will receive a mouse-adapted version of induction therapy currently applied for treatment of MLL-rearranged infant ALL patients. Disclosures No relevant conflicts of interest to declare.

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Role of the Histone Deacetylase Inhibitor Givinostat (ITF2357) in Treatment of CRLF2 Rearranged Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.2534.2534 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2534-2534 ◽

Cited By ~ 1

Author(s):

Angela Maria Savino ◽

Jolanda Sarno ◽

Luca Trentin ◽

Margherita Vieri ◽

Grazia Fazio ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Combined Treatment ◽

Cytokine Receptor ◽

Human Leukemia ◽

Xenograft Models ◽

Stat Pathway

Abstract B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) represents 35% of all cancers in pediatric age group. The cure rate for this disease approaches 90% with current treatment regimens, however only a third of patients with relapse are cured. Therefore, there is an urgent need to focus on subgroups of patients with hallmarks of bad prognosis that could benefit from novel therapeutic approaches. Alterations of Cytokine Receptor-like Factor 2 (CRLF2), a negative prognostic factor in pediatric BCP-ALL, have been identified in up to 10% of patients. However these patients represent half of the high risk Ph-like ALL and of Down Syndrome-associated BCP-ALL. Rearrangements of CRLF2 result in the overexpression of this component of the heterodimeric cytokine receptor for thymic stromal lymphopoietin (TSLP) and is associated with activating mutations of the JAK-STAT pathway. Together these cause hyperactivation of JAK/STAT and PI3K/mTOR signaling. Inhibition of CRLF2/JAK2 signaling has the potential to become a therapeutic targeted intervention for this subgroup of poor prognostic patients. Previous studies have shown that the HDAC inhibitor Givinostat/ITF2357 has potent anti-tumor activity against hematological malignancies, particularly JAK2V617F mutated myeloproliferative neoplasms (MPN) such as polycythemia vera, for which it has already a clinic application and established safety profile. We therefore studied the in vitro and in vivo efficacy of Givinostat in cases with CRLF2 rearrangements. Here we demonstrated that Givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged MHH-CALL4 and MUTZ5 cell lines positive for exon 16 JAK2 mutations. Of note, the observed IC50 values for MHH-CALL4 were lower than those for the SET2 cell line positive control bearing JAK2V617F mutation, both for proliferation (IC50: 0.08±0.05µM vs. 0.14±0.03µM) and apoptosis (IC50: 0.17±0.03µM vs. 0.22±0.04µM). We next investigated the effect of Givinostat on blasts from CRLF2 rearranged BCP-ALL patient samples. For this purpose we developed xenograft models of human CRLF2 rearranged ALL to expand cells from patients and to recapitulate human leukemia in recipient mice. ALL blasts isolated from xenografts were co-cultured on OP9 stroma to perform ex vivo assays. Consistent with our findings in cell lines, Givinostat (0.2µM) reduced the % of live cells (Annexin V/Sytox negative) in all xenografts treated with the drug. In particular, after 72 hours, Givinostat was able to kill up to >90% of blast cells in all xenografts in contrast with the vehicle-treated samples which showed 25-60% of blasts still alive after treatment. The induction of cell death in Givinostat treated primografts was confirmed on primary samples from diagnosis using CyTOF which allowed us to observe that CD10+/CRLF2+ blasts were preferentially killed by the drug whereas CD45 high expressing cells (normal residue) remained unaffected by the treatment. Moreover, at low doses (0.2 µM), Givinostat downregulated genes of the JAK/STAT pathway (STAT5A, JAK2, IL7Rα, CRLF2, BCL2L1 and cMYC) and inhibited the basal and ligand induced signaling, reducing the phoshporylation of STAT5 in all tested primografts (mean fold decrease of pSTAT5: 2.4+0.6). Most importantly, to understand if the transcriptional downregulation of CRLF2 resulted in a functional effect, the downmodulation of CRLF2 protein was observed by flow cytometry (mean fold decrease 3.55+1.38). In vivo, Givinostat significantly reduced engraftment of human blasts in xenograft models of CRLF2 positive BCP-ALL (ranging from 1.9 to 34 fold decrease in bone marrow). Furthermore, Givinostat augmented the effect of chemotherapy in inhibiting proliferation and inducing apoptosis in CRLF2 rearranged cell lines and in primografts, in vitro. After 72 hours, the combined treatment reached 4.6-8.8 fold lower % of remaining viable blasts than chemotherapy alone (6.3-35.3% viable cells in chemotherapy-treated samples vs 1.4-4.3% of combination), 2.5-8.5 fold lower than Givinostat alone (4.3-36.4% vs 1.4-4.3%) and 2.4-13 fold lower than Methyl-prednisolone (5.2-39.1 vs 1-16.3%). In conclusion, Givinostat may represent a novel and effective tool, in combination with current chemotherapy, to treat this difficult to handle subset of ALL and these data strongly argue for the translation of Givinostat in combination with conventional therapy into human trials. Disclosures Davis: Fluidigm, Inc: Honoraria. Nolan:Fluidigm, Inc: Equity Ownership.

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CD52 and CD20 as Potential Targets for Anti-Leukemic Immunotherapy in Adult Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v104.11.4403.4403 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4403-4403

Author(s):

Krystyna Jagoda ◽

Sebastian Giebel ◽

Beata Stella-Holowiecka ◽

Malgorzata Krawczyk-Kulis ◽

Jerzy Holowiecki

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Antigen Expression ◽

Cellular Density ◽

Adult Acute Lymphoblastic Leukemia ◽

Potential Applicability ◽

B Lineage ◽

Anti Cd20 ◽

Cellular Antigen

Abstract CD52 and CD20 are potential targets for immunotherapy of adult acute lymphoblastic leukemia (ALL) with the use of commercially available humanized monoclonal antibodies (MoAbs) (alemtuzumab, rituksimab, etc.). Altough 20% antigen expression is considered diagnostic, it is thought that only patients with >50% and preferably those with >80% leukemic blasts expressing particular antigen are candidates for the treatment with the use of MoAbs. The efficiacy of immunotherapy depends, however, not only on the proportion of lymphoblasts bearing particular marker but also on the cellular antigen density. Therefore, in this study we analyzed both parameters for 29 B-lineage (common/preB n=23, prepreB n=4, B n=2) and six T-lineage (early T n=4, Thymic n=1, matureT n=1) adult ALL patients in order to determine the potential applicability of anti-CD52 and anti-CD20 therapy. METHODS: Samples included only bone marrow aspirates. MoAbs anti CD19 (Immunotech) or anti CD7 (BD) were used for gating lymphoblasts on SSC/FL dotplot. Both tested MoAbs: anti-CD52- R-PE (Caltag, clone CF1D12) and anti-CD20-PE (BD, clone L27) were analyzed in lymphoblasts. The same method (direct staining - three-color fluorescence, flow cytometry - Epics-XL-MCL with System II software, and WinMdi 2.8) was used to compare both antigens. The expression of CD52 and CD20 antigens on blast cells was analyzed as a percentage of positivity (%), and a ratio of the mean intensity fluorescence of lymphoblasts and that of isotypic controls (MIF bl/ctrl). RESULTS: For B-lineage ALL the proportion of patients with CD52 and CD20 positivity was as follows: > 20% positive blasts: 86% vs. 55% (p=0.02), >50% positive blasts: 79% vs. 38% (p=0.003), >80% positive blasts: 48% vs. 24% (p=0.1), respectively. For T-lineage, 2/6 (33%) patients had >50% CD52-positive blasts and none was CD20-positive. Including patients with >50% expression, the ‘relative’ cellular antigen density did not differ for CD52 and CD20: 3.0 (2.2–5.6) vs. 3.9 (1.9–9.5) MIF bl/ctrl (p= NS). In this subgroup the MIF bl/ctr correlated positively with the proportion of blasts expressing CD52 (R Spearman 0.52, p<0.05) whereas the parallel correlation could not be found for CD20 (R Spearman 0.25, p=NS). CONCLUSIONS: The vast majority of adult B-lineage ALL patients may potentailly be treated with anti-CD52 MoAb and less than 50% with anti-CD20 MoAb. However, if CD52- or CD20-positive, the cellular density of the antigen is similar so that the the cytostatic potential of both kinds of immunotherapy could be considered comparable as well. The cellular antigen density of both CD52 and CD20 varies between individual patients, however, in case of CD52 it may be predicted to some extent by the proportion of lymphoblasts bearing the antigen. The anti-leukemic immunotherapy could be considered for a second-line traetment of ALL as well as for in vivo purging before transplantation.

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An Fc-Engineered CD19 Antibody Engages Macrophages and Is Effective in Xenograft Models of Pediatric Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v128.22.277.277 ◽

2016 ◽

Vol 128 (22) ◽

pp. 277-277

Author(s):

Denis M Schewe ◽

Ameera Alsadeq ◽

Gunnar Cario ◽

Simon Vieth ◽

Thomas Valerius ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Antibody Therapy ◽

Patient Specific ◽

Rank Test ◽

Xenograft Models ◽

Overt Leukemia

Abstract Introduction: CD19 antibody therapy may represent an attractive treatment option in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Since conventional CD19 antibodies have failed in clinical trials, different strategies are evaluated to target CD19 more efficiently. Beside the bispecific T cell engager blinatumomab and chimeric antigen receptor T-cells, antibody drug conjugates and antibodies with engineered fragments crystallisable(Fc)for improved effector cell engagement are under investigation. Here, we demonstrate the efficacy of Fc-engineered CD19 antibodies in minimal residual disease (MRD) xenograft models of pediatric BCP-ALL. We further suggest an important contribution of macrophages for this type of therapy. Methods: An Fc-engineered CD19 antibody carrying amino acid mutations S239D/I332E (CD19-DE) and its native CD19-IgG1 variant were generated according to published sequences. CD19-DE was analyzed in patient-derived leukemia xenografts from infants with MLL-rearranged BCP-ALL, which were established by intrafemoral transplantation of 100 cells per animal in NOD-SCID-gamma-/- (NSG) mice lacking a functional lymphatic compartment. CD19-DE was injected intraperitoneally (1 mg/kg on days +1, +3, +6, +10, +13, and every 21 days thereafter; MRD-model). In some experiments leukemia development (defined as >1% peripheral blasts; overt leukemia model) was awaited before CD19-DE was applied alone, or in combination with a regimen mimicking standard induction chemotherapy (Dexamethasone days 1-5, Vincristine day 1 and PEG-Asparaginase day 1 every 28 days). MRD status was determined by analysis of bone marrow DNA for patient-specific immunoglobulin (Ig)-rearrangements and MLL-fusion genes by polymerase chain reaction. In order to test the role of macrophages as effector cells, macrophages were depleted by intraperitoneal injection of liposomal clodronate. In vitro phagocytosis of BCP-ALL primary cells from xenografts was determined by fluorescence microscopy. For that purpose, macrophages were differentiated from human monocytes with macrophage colony-stimulating factor and BCP-ALL cells were labelled with a fluorescent membrane dye. Results: CD19-DE was efficient in prolonging the survival of NSG xenografts of two patients tested in the MRD-model (p = 0.0072 and p = 0.0015, Kaplan-Meier log rank test, Figure A/B). Interestingly, analyses of bone marrow DNA from the surviving mice for two patient specific Ig-rearrangements and the respective MLL-fusion revealed that 4/5 mice were MRD-negative by Ig-rearrangement and 3/5 mice were MRD-negative by MLL-fusion. In order to identify effector mechanisms, antibody therapy was performed in the MRD-model with and without depletion of macrophages. Macrophage depletion in vivo resulted in a reversal of the beneficial effects of CD19-DE as measured by increases in splenic volumes and percentage of human blasts in the bone marrow, suggesting an important role for macrophages in CD19 antibody therapy. CD19-DE was next analyzed for its ability to engage human macrophages in phagocytosis assays with primary BCP-ALL blasts from xenograft mice in vitro. CD19-DE effectively triggered phagocytosis of BCP-ALL cells, whereas a corresponding native CD19 IgG1 antibody did not (ANOVA, p < 0.0001, Figure C; data points indicate results with macrophages from 5 different donors), which emphasizes the importance of Fc-engineering for the efficacy of CD19 antibodies. Finally, therapy with CD19-DE was initiated in the overt leukemia model alone and in combination with chemotherapy. CD19-DE was still efficient in prolonging survival as compared to control animals (p = 0.0003, Figure D), but the effects were less pronounced. Importantly, the combination of antibody therapy and cytoreductive chemotherapy resulted in prolonged survival of 90% of the animals as compared to control animals (p < 0.0001) or animals treated with chemotherapy alone (p = 0.0054; Figure D). Conclusion: These preclinical in vivo data obtained in xenograft models of BCP-ALL suggest a high therapeutic potential of Fc-engineered CD19 antibodies and indicate an important role for macrophages in that context. Administration of Fc-engineered CD19 antibodies in an MRD situation or concomitant application of the antibody and cytoreductive chemotherapy may represent promising approaches in the therapy of pediatric BCP-ALL. Figure Figure. Disclosures Gramatzki: Janssen: Other: Travel/Accommodation/Expenses, Research Funding.

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The Molecular Subtype of Adult Acute Lymphoblastic Leukemia Samples Determines the Engraftment Site and Proliferation Kinetics in Patient-Derived Xenograft Models

Cells ◽

10.3390/cells11010150 ◽

2022 ◽

Vol 11 (1) ◽

pp. 150

Author(s):

Anna Richter ◽

Catrin Roolf ◽

Anett Sekora ◽

Gudrun Knuebel ◽

Saskia Krohn ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Molecular Subtype ◽

Lymphoblastic Leukemia ◽

Clonal Diversity ◽

Model Systems ◽

Leukemic Cells ◽

Patient Derived Xenograft ◽

Molecular Stability ◽

Xenograft Models

In acute lymphoblastic leukemia (ALL), conventional cell lines do not recapitulate the clonal diversity and microenvironment. Orthotopic patient-derived xenograft models (PDX) overcome these limitations and mimic the clinical situation, but molecular stability and engraftment patterns have not yet been thoroughly assessed. We herein describe and characterize the PDX generation in NSG mice. In vivo tumor cell proliferation, engraftment and location were monitored by flow cytometry and bioluminescence imaging. Leukemic cells were retransplanted for up to four passages, and comparative analyses of engraftment pattern, cellular morphology and genomic hotspot mutations were conducted. Ninety-four percent of all samples were successfully engrafted, and the xenograft velocity was dependent on the molecular subtype, outcome of the patient and transplantation passage. While BCR::ABL1 blasts were located in the spleen, KMT2A-positive cases had higher frequencies in the bone marrow. Molecular changes appeared in most model systems, with low allele frequency variants lost during primary engraftment. After the initial xenografting, however, the PDX models demonstrated high molecular stability. This protocol for reliable ALL engraftment demonstrates variability in the location and molecular signatures during serial transplantation. Thorough characterization of experimentally used PDX systems is indispensable for the correct analysis and valid data interpretation of preclinical PDX studies.

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Potent efficacy of combined PI3K/mTOR and JAK or ABL inhibition in murine xenograft models of Ph-like acute lymphoblastic leukemia

Blood ◽

10.1182/blood-2016-05-707653 ◽

2017 ◽

Vol 129 (2) ◽

pp. 177-187 ◽

Cited By ~ 65

Author(s):

Sarah K. Tasian ◽

David T. Teachey ◽

Yong Li ◽

Feng Shen ◽

Richard C. Harvey ◽

...

Keyword(s):

Signal Transduction ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Mtor Inhibition ◽

Key Points ◽

Xenograft Models

Key Points PI3K/mTOR inhibition potently inhibited leukemia proliferation and signal transduction in vivo in human Ph-like ALL xenograft models. Combined PI3K/mTOR and JAK or ABL inhibition was superior to monotherapy in CRLF2/JAK-mutant and ABL/PDGFR-mutant Ph-like ALL models.

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