In vivo monitoring of JAK/STAT and PI3K/mTOR signal transduction inhibition in pediatric CRLF2-rearranged acute lymphoblastic leukemia (ALL).

9506 Background: Therapy intensification for children with B-precursor ALL with high-risk genetic lesions has improved relapse-free survival. CRLF2 rearrangements and JAK2 and IL7RA mutations occur in 10-15% of adult and pediatric ALL patients, most of whom relapse. We and others identified aberrant kinase signatures and perturbed JAK/STAT and PI3K/mTOR signal transduction via in vitro studies of CRLF2-rearranged (CRLF2r) ALLs, suggesting the therapeutic relevance of signal transduction inhibitors (STIs). Our creation of CRLF2r ALL xenograft models has enabled rapid preclinical testing of STIs and measurement of in vivo target inhibition. We hypothesized that inhibition of JAK/STAT and PI3K/mTOR phosphosignaling correlates with therapeutic responses in these models. Methods: NOD/SCID/γc-null (NSG) mice well-engrafted with pediatric ALL samples were treated with the JAK inhibitor ruxolitinib, the mTOR inhibitor sirolimus, or vehicle for 72 hours (for signaling response) or 4 weeks (for therapeutic response). Splenocytes were briefly stimulated ex vivo with thymic stromal lymphopoietin (ligand for CRLF2) and stained with human-specific surface and intracellular phosphoantibodies for multi-parameter phosphoflow cytometry analysis. Results: Ruxolitinib-induced inhibition of phospho (p)-JAK2 and pSTAT5 was most pronounced in non-CRLF2r ALLs with novel JAK2-activating BCR-JAK2 and IL7RA/LNK mutations. Sirolimus potently inhibited pS6 and other PI3K/mTOR pathway phosphoproteins in the CRLF2r r ALLs. PSTAT5 and pS6 inhibition correlated with longer-term ruxolitinib- and sirolimus-induced decreases in ALL cell burden, demonstrating therapeutic responses to STIs. Conclusions: Ruxolitinib inhibited JAK/STAT phosphosignaling and markedly decreased leukemic burden in the JAK2-activating BCR-JAK2 and IL7RA/LNK mutant ALL xenografts. Sirolimus potently inhibited PI3K/mTOR (as well as some JAK/STAT) phosphosignaling and had greater therapeutic efficacy than ruxolitinib in the CRLF2r ALLs. The safety of ruxolitinib and of temsirolimus with cytotoxic chemotherapy are currently being established in Children’s Oncology Group Phase I trials.

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Evaluation of Patient-Derived Xenografts for Modeling Outcome of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.3759.3759 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3759-3759

Author(s):

Abdulmohsen M Alruwetei ◽

Hernan Carol ◽

Rosemary Sutton ◽

Glenn M Marshall ◽

Richard B Lock

Keyword(s):

Gene Expression ◽

Drug Resistance ◽

Clinical Outcome ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Nsg Mice ◽

Relapse Prediction ◽

Pediatric All ◽

Selection Of

Abstract Introduction: Children with acute lymphoblastic leukemia (ALL) are stratified at diagnosis based on molecular/cytogenetic characteristics and their response to initial treatment to receive risk-adapted multi-agent chemotherapy. The majority of ALL patients are stratified as Intermediate Risk (IR) and present with moderate levels of minimal residual disease (MRD<5x104) after receiving induction therapy, although an unacceptably high proportion of these patients relapse. The lack of specific prognostic features makes it difficult to predict the response of IR patients to treatment. The early identification of patients who are destined to relapse would facilitate improvements in tailored treatments for IR ALL patients. Recent progress in the development of patient-derived xenografts (PDXs) in immune-deficient mice represents an opportunity to improve relapse prediction in ALL patients. The aims of this study were to: (1) optimize the engraftment conditions of IR pediatric ALL samples to predict patient response to treatment; and, (2) to assess the development and mechanisms of therapy-induced drug resistance. Methods: Two pairs of IR pediatric ALL patients were matched based on clinical and genetic features, except that one patient from each pair relapsed early while the other remains relapse-free (ALL-Rel and ALL-CR1, respectively). Three parameters were varied in establishing PDXs by inoculating one million bone marrow (BM) derived biopsy cells collected at diagnosis into groups of 4 mice: (1) mouse strain (NOD/SCID vs. NSG); (2) site of inoculation (intravenous vs. intra-femoral); and (3) early treatment of mice with a 2-week induction chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Leukemia engraftment was monitored weekly based on the proportion of human versus mouse CD45+ cells in the murine PB, and the median times to engraftment were compared according to patient outcome. The median time to engraft was also compared between the VXL-treated and non-treated groups. PDXs harvested from mice were compared for ex vivo sensitivity to single agent vincristine, dexamethasone and L-asparaginase. PDX gene expression profiles were also compared to identify pathways associated with evasion of VXL treatment in vivo. Results: The efficiency of engraftment was greater for NSG mice (29/32 mice engrafted) versus NOD/SCID mice (20/32 mice), and primary ALL cells also engrafted significantly faster in NSG mice (median time to engraft 71.1 days) compared with NOD/SCID mice (83.5 days) (P < 0.01), with no apparent difference associated with clinical outcome. Intrafemoral inoculation did not improve the efficiency or speed of engraftment compared with intravenous inoculation, nor predicted clinical outcome. However, PDX responses to VXL induction chemotherapy reflected the clinical outcome of the patients from whom they were derived; those derived from the 2 ALL-Rel patients exhibited in vivo drug resistance (leukemia growth delay of 1 and 6.2 days) compared with those derived from the 2 ALL-CR1 patients (34.7 and >119.8 days). Further, ex vivo analysis showed that the PDXs derived from the ALL-Rel patients exhibited resistance to vincristine or L-asparaginase compared with those derived from the ALL-CR1 cases. Moreover, the in vivo VXL treatment of an ALL-CR1 PDX resulted in selection of cells that exhibited vincristine resistance. Gene expression profiling revealed significant up-regulation of microtubule associated proteins (MAPs) and tubulin isotypes (alpha and beta) in vincristine-resistant PDXs. Genes that were significantly upregulted in vincristine resistant PDXs with a false discovery rate (FDR) < 0.05 and P value < 0.02 include TUBB6, TUBA1A, TUBA1B, MAP1S, TUBA3D and TBCA. The increased expression of genes that affect microtubule functions suggest that changes in microtubule dynamics and/or stability led to decreased sensitivity to antimicrotubule agents. Conclusions: In vivo selection of PDXs with an induction-type regimen of chemotherapeutic drugs may lead to improved relapse prediction and identify novel mechanisms of drug resistance in IR pediatric ALL. Support: Steven Walter Foundation; NHMRC Australia, APP1057746 Disclosures No relevant conflicts of interest to declare.

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The Regulation of β-arrestin1 in Leukemia Initiating Cells of Children B-Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.3538.3538 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3538-3538

Author(s):

Lin Zou ◽

Shan Liu ◽

Yi Shu ◽

Ru Qin ◽

Kang Li ◽

...

Keyword(s):

Dna Methylation ◽

Mrna Expression ◽

Promoter Region ◽

Lymphoblastic Leukemia ◽

The Self ◽

Mrna Expression Level ◽

Self Renewal ◽

Nsg Mice

Abstract Background Leukemia is the most common malignant tumor in children under 15 years old. The main subtype of children leukemia is acute lymphoblastic leukemia (ALL), and B-lineage ALL (B-ALL) accounts for approximately 70%. The leukemia-initiating cells (LICs) are cancer stem cells with long-term repopulating potential and propagation ability, to maintain the leukemia cell phenotype, and possess leukemia-initiating activity. However, the regulation of LICs for the leukemia progression is poorly understood. The multifunctional scaffold proteins β-arrestins are proven to mediate H4 acetylation and gene expression. And β-arrestin2 is found to regulate the initiation and progression of chronic myeloid leukemia (CML). However, the role of β-arrestin1 in B-ALL is still unknown. Our preliminary data showed that both the high expression of β-arrestin1 and high proportion of CD34+CD38- cells are positively correlated with risk stratification and poor prognosis of childhood B-ALL. And β-arrestin1 binds with EZH2 to increase BCR/ABL H4 acetylation and thus promotes CML cell progression in vitro and in vivo. The aim of study is to investigate the essential function of β-arrestin1 in LICs from B-ALL. Materials and Methods The bone marrow (BM) and periphery blood (PB) of children B-ALL patients were collected, isolated and identified LICs by Magnetic-activated cell sorting (MACS) and flow cytometry. The total RNA and protein were purified for gene and protein expression by real-time RT-PCR and Western blot. The leukemia cells (LICs, Raji, and Reh) of β-arrestin1 depletion were constructed by transient or stable screening si-β-arrestin1 (siβ1) lentivirus vector. The serial cell colony formation and NSG mice survival analysis was measured the LICs self-renewal ability. The CCK8 and MTS assays were used to detect the cell proliferation, and annexin V-FITC and PI staining for cell apoptosis. The DNA methylation of gene promoter region was detected by methylation-specific PCR and the methltransferase activity by ELISA. The telomere length was indicated by Southern blot and FISH, and telomerase activity by TRAP. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay were applied to explain gene transcription. Student’s t test and Log-Rank test were used in the corresponding statistical significance and P<0.05 were considered significant. All the statistical analysis was performed using the GraphPad Prism (Version 5.0) software packages and SPSS 17.0. Results The expression of β-arrestin1 was elevated in LICs from B-ALL patients, and the high level of β-arrestin1 was negatively correlated with the survival of these patients. Further study showed that the loss of β-arrestin1 in B-ALL LICs attenuates their self-renewal capacity and promotes their senescence in vitro and in vivo. The mRNA expression level of β-arrestin1 is negatively correlated with that of PTEN in LICs. Moreover, the DNA methylation of the PTEN promoter region, the activity and the expression of DNMTs were enhanced in the LICs. The inhibition of DNMT1 activity impaired the self-renewal and increased the expression of PTEN of LICs. In addition, depletion of β-arrestin1 significantly decreased DNMT1 activity and PTEN methylation, and consistently increased PTEN expression in LICs. For B-ALL cell senescence, the mRNA expression level of β-arrestin1 is negatively related with the length of telomere, positively related with the activity of telomerase and the mRNA expression of hTERT in B-ALL LICs and engrafted NSG mice. Moreover, the weakened effect of β-arrestin1 on telomere, telomerase and the gene of hTERT were observed by injected the inhibitor of telomerase in leukemic mice. In addition, depletion of β-arrestin1 significantly decreased the binding of SP1 to the promoter of hTERT and thus reduced the transcription of hTERT in B-ALL Raji and Reh cells. Furthermore, β-arrestin1 interacted with P300 to bind with SP1 in the -104bp to -113bp of hTERT core promoter region in B-ALL cells. Conclusions β-arrestin1 could regulate the self-renewal and senescence of LICs from B-ALL, by partially mediating DNMT1 activity and hTERT transcription respectively, indicating that β-arrestin1 is a potential therapeutic target for B-ALL. Disclosures No relevant conflicts of interest to declare.

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Targeted Cancer Therapy in High-Risk Pediatric Leukemia Using Global Phosphotyrosine Profiling

Blood ◽

10.1182/blood.v124.21.969.969 ◽

2014 ◽

Vol 124 (21) ◽

pp. 969-969

Author(s):

Sibasish Dolai ◽

Keith CS Sia ◽

Alissa K Robbins ◽

Ling Zhong ◽

Sue Heatley ◽

...

Keyword(s):

High Risk ◽

B Cell ◽

Tyrosine Phosphorylation ◽

Lymphoblastic Leukemia ◽

Cell Precursor ◽

Pediatric Leukemia ◽

Pediatric All ◽

Quantitative Tool

Abstract Introduction: While cure rates for children with acute lymphoblastic leukemia (ALL) are approaching 90% with conventional chemotherapeutic regimens, certain high-risk patient subsets such as early T-cell precursor ALL (ETP-ALL) and Philadelphia Chromosome-like (Ph-like) ALL have an aggressive disease profile and poor prognosis. More recently whole genome and transcriptome sequencing of these high-risk subtypes have revealed several activating gene fusions, alterations and mutations that could result in constitutively activated tyrosine kinases (TKs). Activated TKs are then capable of phosphorylating downstream substrates and impacting several key signaling pathways, resulting in increased cell survival, proliferation and differentiation. Further, the highly heterogeneous nature of these subtypes, along with activating fusions/mutations, makes them refractory to standard chemotherapy. Consequently, there is an urgent need to develop tailored therapeutic strategies for the treatment of these high-risk ALL subtypes. Recent advances in mass-spectrometry and the use of anti-phosphotyrosine antibodies for enrichment of tyrosine phosphorylated peptides have greatly facilitated characterization of the global tyrosine phosphorylation state in cancer cells and identified activated TKs that could be therapeutically targeted. Here we present the first study to quantitatively profile TK activity in xenografted patient biopsies of high-risk pediatric ALL. Methods: In this study, we have established an MS-based phosphotyrosine profiling approach in patient derived xenografts (PDXs) of high-risk pediatric ALL patients and integrated it with a spike-in SILAC quantitative tool to identify and quantify dysregulated TK activity across 16 PDXs. We further extended our study on markedly altered tyrosine phosphorylation in 4 PDXs to assess the therapeutic potential of specific TK inhibitors (TKIs). Immunoblots were performed to validate activated sites and their dephosphorylation upon TKI treatment. RT-PCR and Exome sequencing was carried out to detect novel fusion partners and point mutation sites to validate the activated TK profiles in these PDXs. In vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. PDXs were established from ETP-ALL, Ph-like ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice. Engraftment and in vivo drug responses were assessed by enumeration of the proportion of human versus mouse CD45+cells in the murine PB. Results: Using a quantitative phosphotyrosine profiling method in 16 PDXs, we mapped close to 1900 class I phosphosites with >0.75 localization probability and 99% confidence, of which 1394 tyrosine phosphorylated sites had a heavy SILAC partner that allowed quantification. Such profiling could accurately classify the leukemias into either T or B-cell lineages with the high-risk ETP and Ph-like ALL clustering as a distinct group. In particular, PDXs with activated tyrosine phosphorylation profiles of ABL1, FLT3 and JAK were targeted with commercially available TKIs both in vitro and in vivo. Subsequent analysis to investigate the aberrant ABL1 and FLT3 signaling showed a NUP214-ABL1 translocation unique to BCP-ALL in one PDX, and a novel Y572S FLT3 mutation in another. Importantly, using a pre-clinical in vivo xenograft model, the activated JAK-STAT signaling observed in one ETP-ALL PDX was targeted with the JAK1/2 inhibitor, ruxolitinib, leading to a significant decrease in the leukemic blast population in the murine PB. Aberrant ABL1 kinase signaling indicated dasatinib treatment in a Ph+-ALL PDX and a PDX with high phospho-ABL1 (harboring the NUP214-ABL1 translocation), and a complete response and significant progression delay, respectively, were achieved in vivo. Similarly, the uniquely activated FLT3 in one PDX (Y572S mutation) correlated with an in vivoobjective response to the multi-kinase inhibitor sunitinib. Conclusions: This study demonstrates the direct application of an unbiased and quantitative tool to identify aberrant TK signaling in high-risk ALL PDXs and highlights its potential to identify tractable drug targets. This research was supported by NCI NO1CM42216 and by the Australian National Health and Medical Research Council. Disclosures No relevant conflicts of interest to declare.

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The Pro-Tumorigenic Vascular Niche Sustains the T-Cell Acute Lymphoblastic Leukemia Phenotype and Fosters Resistance to Therapy

Blood ◽

10.1182/blood.v128.22.279.279 ◽

2016 ◽

Vol 128 (22) ◽

pp. 279-279

Author(s):

Filomena Di Giacomo ◽

Xujun Wang ◽

Danilo Fiore ◽

Lorena Consolino ◽

Jude Phillip ◽

...

Keyword(s):

High Risk ◽

Cell Lines ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Serum Cytokine ◽

Nsg Mice ◽

Cell Acute Lymphoblastic Leukemia ◽

Free Media

Abstract Introduction. T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malignancy associated with a high risk of treatment failure. Efforts to improve outcomes have focused on underlying genetic defects. However, new evidence suggests that the microenvironment can foster drug resistance/relapses. Identification of factors that contribute to microenvironment-mediated chemo-refractoriness remains an important challenge. Here, we sought to construct an in vitro platform to dissect tumor-host interactions and to optimize drug treatments using Patient-Derived Tumor Xenograft models (PDTX) of high risk adult T-ALL and engineered human endothelial cells. Methods. T-ALL PDTX were established and serially passaged in NSG mice. Engraftment was monitored by flow cytometry of peripheral blood and/or MRI. Mice were sacrificed and leukemic cells were harvested from the spleen/bone marrow. To determine the ex vivo growing conditions, we first cultured a panel of 8 "bona fide" T-ALL cell lines and 11 PDTX cells alone in complete RPMI 20% FCS supplemented with IL2, IL12, IL15 and IL7; or co-cultured with human E4-ORF1 endothelial cells (ECs) without ILs in complete RPMI 20% FCS or serum/cytokine-free media. CDK4/6, MEK, PI3K and JAK inhibitors were used at 0.1 and 1 µM alone and in combination. Cell titer glo, cell titer blue, Annexin-V and S-cell cycle analysis were used as readouts. Total RNA from cells before and after co-culture was extracted for paired-end RNA sequencing on an Illumina HiSeq2500. Results. To study the supporting role of ECs, we first co-cultured ECs with T-ALL cell lines in vitro (serum/cytokine free co-culture) and showed that ECs could reproducibly sustain the viability of 3/8 cell lines (Loucy, KOPTK1, P12 Ichikawa) serum/cytokine-free media. A partial rescue was seen with 3 additional lines (HPB-ALL, CCRF-CEM, CUTLL1), while 2 (KE37, DND41) underwent massive cell death. We next tested whether either ILs or CXCL12 could provide anti-apoptotic signals and demonstrated that KOPTK1 and Loucy were only partially rescued by IL15 or CXCL12. Conversely, IL7, although capable of inducing a robust upregulation of pSTAT5, had no effect (CCRF-CEM and CUTLL1). We then characterized 11 PDTX from 15 high-risk adult T-ALL patients. All PDTX were serially propagated and caused T-ALL in subsequent NSG mice (massive spleen and bone marrow infiltration with extensive paravertebral mass associated with paralysis and multi-organ involvement). Genomic analysis (RNA-seq) demonstrated a high concordance between primary (pre-implant) and PDTX samples. All of them were extensively studied ex vivo, demonstratingthat T-ALL PDTX cells could only survive in ILs supplemented media, even better if enriched of growth factors and supplements for the expansion of human hematopoietic cells. However, when PDTX cells were treated with targeting compounds they all underwent massive apoptosis. Conversely, individual PDTX T-ALL could be selectively rescued by ECs, allowing the construction of individual drug response profile. To extend these data, 7 PDX T-ALL samples were screened against a 430-targeted compound library in supplemented RPMI or Stem Span media. Results indicated differential cell killing and gain (NFKB, BTK) and loss (TP-53, IGF-1R) of targets. Conclusions. These data clearly demonstrate a key role of aberrantly activated vascular niche in T-ALL cell maintenance and drug resistance. We envisage that drug screening of EC+T-ALL will lead to the identification of actionable targets in each individual patient. Our report supports the potential for future personalized curative strategies aimed at targeting both tumor cells and host tissue supporting niche elements disrupting pro-tumorigenic signals within leukemia cell niches. Disclosures Foà: Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy; Pfizer: Speakers Bureau; Ariad: Speakers Bureau. Rafii:Angiocrine Bioscience: Equity Ownership, Other: Non-paid consultant.

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Inhibiting PIM-1 Is Effective in Vitro and in Vivo against ALL: A Novel Mechanistic and Potentially Clinically Relevant Druggable Target.

Blood ◽

10.1182/blood.v112.11.1922.1922 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1922-1922

Author(s):

Valerie I. Brown ◽

Cecilia Sheen ◽

Jessica Hulitt ◽

Theresa Ryan ◽

Laura DiNardo ◽

...

Keyword(s):

Lymphoblastic Leukemia ◽

Acquired Resistance ◽

Mtor Inhibitors ◽

In Vivo Model ◽

Mrna Levels ◽

Downstream Target ◽

Signal Transduction Inhibitors ◽

Survival Studies

Abstract The outcome for patients with acute lymphoblastic leukemia (ALL) has improved greatly over the past three decades. However, the prognosis remains dismal for those with relapsed or refractory ALL despite intensified therapy. Biologically targeted agents, such as signal transduction inhibitors (STIs) have shown promise in treating leukemia. We have reported that mTOR inhibitors (MTIs) such as rapamycin (rap), RAD-001, and CCI- 779 show activity in models of murine and human ALL. However, acquired resistance to STIs remains a concern. Furthermore, the presence of cytokines such as IL-7 and TSLP can promote survival, induce STAT5 phosphorylation, and reverse the inhibitory effects of MTIs in ALL cells. We hypothesize that IL-7-mediated signaling promotes ALL cell survival and potentially contributes to MTI resistance by upregulating alternative survival pathways, such as the JAK/STAT pathway. We have evaluated the effects of inhibiting PIM-1 kinase, a known downstream target of STAT5. Using the PIM-1 inhibitor SGI- 1776 (generously provided by SuperGen, Inc.), we have found that SGI-1776 profoundly inhibited proliferation in vitro, with an IC50 of approximately 1 mM for murine and 2.5 mM for human ALL cell lines. Greater than 90% inhibition was seen at concentrations of 5 and 10 mM in murine and human ALL lines, respectively. Furthermore, a combination of 1 mM SGI-1776 and 1 ng/ml rap resulted in further inhibition than either agent alone. Because PIM-1 is regulated at the transcription level, we measured changes in PIM-1 specific mRNA levels via real time PCR after 24 hour treatment with combinations of SGI-1776, rap and IL-7 (2 ng/ml). As seen in the Table, in each treatment condition SGI-1776 significantly decreased PIM-1 mRNA. As expected, IL-7 increased PIM-1 expression. Interestingly, inhibition of mTOR signaling via rap also resulted in an apparent compensatory increase in PIM-1 mRNA, which was in turn antagonized by SGI-1776. TABLE: fold change in PIM-1 mRNA by RT-PCR Untreated SGI-1776 IL7 IL7+SGI Rap Rap+SGI Rap+IL7 Rap+IL7+SGI 1 0.1 17 1 3 0.4 22 6.5 To evaluate SGI-1776 in a clinically relevant in vivo model, NOD/SCID mice xenografted with human primary ALL cells from several samples were treated with SGI-1776 alone, SGI-1776 + rap or drug vehicle. SGI-1776 (200 mg/kg/dose daily x 5 per week by gavage) alone or with rap decreased in vivo tumor proliferation over time as compared to untreated mice. At this dose of SGI-1776, the treated mice exhibited significant side effects, including weight loss, hunched appearance with scruffy coats, decreased appetite and decreased activity. Because of this toxicity, we were not able to detect a difference in survival as a result of observed decreases in ALL burden; however these toxicities were alleviated with a reduction of SGI-1776 to 100 mg/kg/dose, and survival studies at the better-tolerated dose are ongoing. These data show that, alone and in combination with rapamycin, the PIM-1 inhibitor SGI-1776 demonstrates activity in vitro and in vivo against human ALL. Together these data suggest that PIM-1 activation can act as a mechanism of cytokine mediated MTI resistance, making PIM-1 an attractive therapeutic target for ALL.

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Treatment of Ex Vivo Expanded NK Cells with Daratumumab F(ab')2 Fragments Protects Adoptively Transferred NK Cells from Daratumumab-Mediated Killing and Augments Daratumumab-Induced Antibody Dependent Cellular Toxicity (ADCC) of Myeloma

Blood ◽

10.1182/blood.v126.23.4244.4244 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4244-4244 ◽

Cited By ~ 6

Author(s):

Elena Cherkasova ◽

Luis Espinoza ◽

Ritesh Kotecha ◽

Robert N. Reger ◽

Maria Berg ◽

...

Keyword(s):

Nk Cells ◽

Adoptive Transfer ◽

Nk Cell ◽

Ex Vivo ◽

Cell Killing ◽

Cellular Toxicity ◽

Myeloma Cells ◽

Nsg Mice

Abstract Daratumumab is a fully humanized monoclonal antibody (IgG1) that targets CD38 expressed on myeloma cells. Daratumumab kills myeloma cells through antibody dependent cellular toxicity (ADCC), compliment dependent cytotoxicity (CDC), and antibody dependent phagocytosis (ADCP). In early clinical trials, daratumumab has showed significant anti-myeloma activity in patients with treatment refractory disease. In vivo, daratumumab has been found to induce NK cell lymphopenia of unclear etiology. We found that NK cells isolated from the peripheral blood of healthy and cancer patients expressed variable surface levels of CD38 (Fig. 1A). Further, surface expression of CD38 increased substantially when NK cells underwent ex vivo cytokine activation by culturing cells overnight in IL-2 containing media or ex vivo expansion using irradiated EBV-LCL feeder cells (Fig. 1B). Remarkably, daratumumab induced apoptosis of expanded NK cells in a dose dependent manner, with substantial NK cell apoptosis occurring within 2 hours following in vitro exposure to daratumumab at a concentration of 1 and 10 ug/ml (Fig. 1C). Further, adoptive transfer of ex vivo expanded human NK cells into NSG mice that had been pre-treated with daratumumab showed daratumumab induced NK cell killing in vivo: the numbers of NK cells isolated from the lungs, blood, spleen and bone marrow of NSG mice 24 hours after infusion of expanded human NK cells was reduced by 90% in mice that were pretreated with 1 mg/kg of daratumumab i.p. compared to controls that had not received the antibody (Fig. 1D). In vitro experiments showed NK cell killing by daratumumab occurred as a consequence of ADCC and was dependent on NK cell CD16 expression; when CD56+ NK cells were sorted by FACS into CD16 positive and negative populations, only NK cells expressing CD16 were killed by daratumumab, with no effect on NK cell viability occurring in the CD16- NK cell. Further, we observed that NK cells obtained from donors who have high affinity FCgR3 as a consequence of a single nucleotide polymorphism in the FCGR3A gene resulting in an amino acid substitution at position 158 (F158V) in CD16 were more sensitive to daratumumab killing compared to NK cells isolated from donors carrying the low affinity CD16 polymorphism. Although NK cell counts and NK reduction in peripheral blood and bone marrow were not associated with daratumumab clinical response in myeloma studies, NK cells play an important role in mediating antitumor responses through ADCC following mAb therapy. In this regard, combining mAb therapy with adoptive transfer of ex vivo expanded NK cells could be utilized as a strategy to potentiate the antitumor effects of mAbs. To overcome daratumumab-mediated killing of adoptively transferred NK cells in daratumumab-treated patients, we blocked CD38 on the surface of NK cells by pretreating them with daratumumab F(ab')2 fragments. The F(ab')2 fragments that were generated using pepsin cleavage of daratumumab were confirmed to bind and block the CD38 epitope expressed on NK cells. Importantly, these F(ab')2 fragments remained bound to the surface of NK cells for at least 96 hours, did not induce NK cell apoptosis, protected NK cells from daratumumab-mediated NK cell killing, and bolstered their tumor cytotoxicity against daratumumab-treated myeloma targets. In vitro experiments showed NK cell tumor cytotoxicity vs myeloma cells in daratumumab-containing media was significantly higher by NK cells that had CD38 blocked with F(ab')2 fragments compared to unblocked controls (Fig. 1E). Importantly, pretreatment with daratumumab F(ab')2 fragments also protected human NK cells from daratumumab-mediated killing in vivo; expanded NK cells pretreated with F(ab')2 fragments prior to adoptive transfer into NSG mice that had been treated with daratumumab were detectable at significantly higher numbers in the blood compared to untreated NK cell controls (Fig. 1F). Conclusion: Expression of CD38 on activated NK cells makes them susceptible to killing by daratumumab, which could compromise the ability of adoptively transferred NK cells to bolster ADCC following treatment with this mAb. Pretreatment of ex vivo expanded NK cells with daratumumab F(ab')2 fragments protects cells from daratumumab-mediated killing, potentially offering a strategy to augment the anti-tumor effects of adoptively transferred NK cells in myeloma patients that have received daratumumab treatment. Disclosures No relevant conflicts of interest to declare.

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JAK/STAT pathway inhibition sensitizes CD8 T cells to dexamethasone-induced apoptosis in hyperinflammation

Blood ◽

10.1182/blood.2020006075 ◽

2020 ◽

Vol 136 (6) ◽

pp. 657-668 ◽

Cited By ~ 7

Author(s):

Lauren K. Meyer ◽

Katherine C. Verbist ◽

Sabrin Albeituni ◽

Brooks P. Scull ◽

Rachel C. Bassett ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Immune System Activation ◽

Stat Signaling ◽

Induced Apoptosis ◽

Stat Pathway

Abstract Cytokine storm syndromes (CSS) are severe hyperinflammatory conditions characterized by excessive immune system activation leading to organ damage and death. Hemophagocytic lymphohistiocytosis (HLH), a disease often associated with inherited defects in cell-mediated cytotoxicity, serves as a prototypical CSS for which the 5-year survival is only 60%. Frontline therapy for HLH consists of the glucocorticoid dexamethasone (DEX) and the chemotherapeutic agent etoposide. Many patients, however, are refractory to this treatment or relapse after an initial response. Notably, many cytokines that are elevated in HLH activate the JAK/STAT pathway, and the JAK1/2 inhibitor ruxolitinib (RUX) has shown efficacy in murine HLH models and humans with refractory disease. We recently reported that cytokine-induced JAK/STAT signaling mediates DEX resistance in T cell acute lymphoblastic leukemia (T-ALL) cells, and that this could be effectively reversed by RUX. On the basis of these findings, we hypothesized that cytokine-mediated JAK/STAT signaling might similarly contribute to DEX resistance in HLH, and that RUX treatment would overcome this phenomenon. Using ex vivo assays, a murine model of HLH, and primary patient samples, we demonstrate that the hypercytokinemia of HLH reduces the apoptotic potential of CD8 T cells leading to relative DEX resistance. Upon exposure to RUX, this apoptotic potential is restored, thereby sensitizing CD8 T cells to DEX-induced apoptosis in vitro and significantly reducing tissue immunopathology and HLH disease manifestations in vivo. Our findings provide rationale for combining DEX and RUX to enhance the lymphotoxic effects of DEX and thus improve the outcomes for patients with HLH and related CSS.

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Combinatorial immunotherapy of N-803 (IL-15 superagonist) and dinutuximab with ex vivo expanded natural killer cells significantly enhances in vitro cytotoxicity against GD2+ pediatric solid tumors and in vivo survival of xenografted immunodeficient NSG mice

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-002267 ◽

2021 ◽

Vol 9 (7) ◽

pp. e002267

Author(s):

Yaya Chu ◽

Gaurav Nayyar ◽

Susiyan Jiang ◽

Jeremy M. Rosenblum ◽

Patrick Soon-Shiong ◽

...

Keyword(s):

Solid Tumors ◽

Nk Cell ◽

Ex Vivo ◽

In Vitro Cytotoxicity ◽

Event Free Survival ◽

Free Survival ◽

Nsg Mice ◽

Ifn Γ

BackgroundChildren with recurrent and/or metastatic osteosarcoma (OS), neuroblastoma (NB) and glioblastoma multiforme (GBM) have a dismal event-free survival (<25%). The majority of these solid tumors highly express GD2. Dinutuximab, an anti-GD2 monoclonal antibody, significantly improved event-free survival in children with GD2+ NB post autologous stem cell transplantation and enhanced natural killer (NK) cell-mediated antibody-dependent cell cytotoxicity. Thus, approaches to increase NK cell number and activity, improve persistence and trafficking, and enhance tumor targeting may further improve the clinical benefit of dinutuximab. N-803 is a superagonist of an interleukin-15 (IL-15) variant bound to an IL-15 receptor alpha Su-Fc fusion with enhanced biological activity.MethodsThe anti-tumor combinatorial effects of N-803, dinutuximab and ex vivo expanded peripheral blood NK cells (exPBNK) were performed in vitro using cytoxicity assays against GD2+ OS, NB and GBM cells. Perforin and interferon (IFN)-γ levels were measured by ELISA assays. Multiple cytokines/chemokines/growth factors released were measured by multiplex assays. Human OS, GBM or NB xenografted NOD/SCID/IL2rγnull (NSG) mice were used to investigate the anti-tumor combinatorial effects in vivo.ResultsN-803 increased the viability and proliferation of exPBNK. The increased viability and proliferation are associated with increased phosphorylation of Stat3, Stat5, AKT, p38MAPK and the expression of NK activating receptors. The combination of dinutuximab and N-803 significantly enhanced in vitro cytotoxicity of exPBNK with enhanced perforin and IFN-γ release against OS, GBM and NB. The combination of exPBNK+N-803+dinutuximab significantly reduced the secretion of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), platelet-derived growth factor-BB (PDGF-BB), and stem cell growth factor beta (SCGF-β) from OS or GBM tumor cells. Furthermore, OS or GBM significantly inhibited the secretion of regulated on activation, normal T cell expressed and presumably secreted (RANTES) and stromal cell-derived factor-1 alpha (SDF-1α) from exPBNK cells (p<0.001) but significantly enhanced monokine induced by gamma interferon (MIG) secretion from exPBNK cells (p<0.001). N-803 combined with dinutuximab and exPBNK cells significantly extended the survival of OS, GBM or NB xenografted NSG mice.ConclusionsOur results provide the rationale for the development of a clinical trial of N-803 in combination with dinutuximab and ex vivo exPBNK cells in patients with recurrent or metastatic GD2+ solid tumors.

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Targeting Telomere Biology in Acute Lymphoblastic Leukemia

International Journal of Molecular Sciences ◽

10.3390/ijms22136653 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6653

Author(s):

Axel Karow ◽

Monika Haubitz ◽

Elisabeth Oppliger Leibundgut ◽

Ingrid Helsen ◽

Nicole Preising ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

High Risk ◽

T Lymphocytes ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Genetic Alterations ◽

Adult Patients ◽

Pediatric All ◽

B And T Lymphocytes

Increased cell proliferation is a hallmark of acute lymphoblastic leukemia (ALL), and genetic alterations driving clonal proliferation have been identified as prognostic factors. To evaluate replicative history and its potential prognostic value, we determined telomere length (TL) in lymphoblasts, B-, and T-lymphocytes, and measured telomerase activity (TA) in leukocytes of patients with ALL. In addition, we evaluated the potential to suppress the in vitro growth of B-ALL cells by the telomerase inhibitor imetelstat. We found a significantly lower TL in lymphoblasts (4.3 kb in pediatric and 2.3 kb in adult patients with ALL) compared to B- and T-lymphocytes (8.0 kb and 8.2 kb in pediatric, and 6.4 kb and 5.5 kb in adult patients with ALL). TA in leukocytes was 3.2 TA/C for pediatric and 0.7 TA/C for adult patients. Notably, patients with high-risk pediatric ALL had a significantly higher TA of 6.6 TA/C compared to non-high-risk patients with 2.2 TA/C. The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. These results suggest that TL reflects clonal expansion and indicate that elevated TA correlates with high-risk pediatric ALL. In addition, telomerase inhibition induces apoptosis of B-ALL cells cultured in vitro. TL and TA might complement established markers for the identification of patients with high-risk ALL. Moreover, TA seems to be an effective therapeutic target; hence, telomerase inhibitors, such as imetelstat, may augment standard ALL treatment.

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Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Blood ◽

10.1182/blood-2021-149693 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 194-194

Author(s):

Pamela Wong ◽

Carly C. Neal ◽

Lily Chang ◽

Julia A Wagner ◽

Melissa M. Berrien-Elliott ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

T Box ◽

Nsg Mice ◽

Healthy Donors ◽

And Function

Abstract Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear. We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology. Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p < 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo. To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (<30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p<0.01, Figure C), blood (6-fold decrease, p<0.01), and liver (4-fold decrease, p<0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p<0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p<0.05), and IFN-γ production was reduced (p<0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E). Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. Figure 1 Figure 1. Disclosures Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.

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