Spontaneous Leukemia Research Articles

Abstract 1033: Adult B-cell lymphocytic leukemia associated with osteolysis but normocalcemia and normal hematologic parameters - rare case or influenced by mifepristone treatment

Abstract Acute B-cell lymphoblastic leukemia (All) is much less common in adults than children and is much less common to present with bone pain. Extremely rare, but precedented, is the absence of hematologic abnormalities or lymphadenopathy. In some of the rare cases early diagnosis was achieved because hypercalcemia and bone pain were related to ectopic production of parathyroid-related peptide with consequent hypercalcemia and bone pain from hyperparathyroidism. However, there has been a case report in a 37-year-old male with B-cell lymphoblastic leukemia with osteolytic lesions, normal parathyroid hormone levels, and normal hematologic parameters despite >80% of the bone marrow replaced with blast cells. We present another case of a 55-year-old woman presenting with B cell ALL, bone pain and perfectly normal hematologic parameters despite 95% of the marrow replaced with blast cells. Her pain began 2 ½ months prior to the bone biopsy which was done because of the finding of osteolytic lesions on MRI. Her first bone marrow evaluation was performed 1 ½ months later. Related to further delays in even getting an appointment with the proper oncologist she was started on mifepristone 200mg daily to hopefully help delay a hematologic ALL crisis in view of 95% blast cells on biopsy. After 1 month of mifepristone therapy her CBC was still perfectly normal before standard chemotherapy was started. Mifepristone was started because of its effectiveness in improving length and quality of life in patients with a variety of advanced cancers without any more treatment options coupled with its beneficial effect in controlled studies with spontaneous leukemia in mice. Furthermore, the target for mifepristone, the immunomodulatory protein known as the progesterone induced blocking factor (PIBF), was found to be highly expressed by multiple human leukemia cell lines and was suppressed by mifepristone. The most common clinical state found in patients with advanced cancers following treatment with mifepristone is to stabilize the disease and inhibit further spread. The possibility exists that the failure to progress to typical pancytopenia after 95% of the bone marrow was replaced by blast cells was related to the mifepristone treatment. However, the possibility also exists that some unknown factor inhibiting this progression was operative as in some other very rare reported cases. With the possibility of a beneficial effect of treatment, mifepristone, an extremely well tolerated oral agent, could be considered for treating ALL when no other reasonable treatment options are available, e.g., a patient not likely to survive standard therapy or failure to respond to traditional chemotherapy. Even in this patient, if it takes more than 1 month to induce remission, her 40% 5-year survival will even be lower so one could consider maintenance therapy with mifepristone. Citation Format: Jerome H. Check. Adult B-cell lymphocytic leukemia associated with osteolysis but normocalcemia and normal hematologic parameters - rare case or influenced by mifepristone treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1033.

The Role of Kdm6a in Malignant Hematopoiesis

Our preliminary studies identified recurrent, somatic mutations in Kdm6a in a mouse model of acute promyelocytic leukemia (APL). Putative inactivating mutations of Kdm6a (primarily small deletions) occurred in approximately 50% of mouse APL samples, making these the most common acquired progression events in this model. We found somatic mutations of KDM6A in 4 of 200 acute myeloid leukemia (AML) samples. However, in a cohort of 42 patients with APL, we identified no somatic alterations in KDM6A . To better understand the role of Kdm6a in malignant hematopoiesis, we intercrossed our Kdm6a conditional knockout (KO) x Vav1 - Cre mice with a “knock-in” mouse model of APL (Ctsg-PML-RARA mice). To determine whether Kdm6a inactivation cooperates with PML - RARA, we performed a tumor watch that included all genotypes of Kdm6a conditional KO female and male triple cross mice. After 18 months of observation, we found that the latency of APL was significantly decreased in all three cohorts of Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (Figure 1). This finding was most pronounced in female homozygous Kdm6a KO triple cross mice, which had a median survival of 161 days versus 365 days in the control cohort (P < 0.01 by log-rank test). The Kdm6a -null mice developed the typical phenotype of mouse APL, including splenomegaly, myeloproliferation and leukemic cells that coexpressed Gr-1 and Kit. The triple cross APL cells remained sensitive to all- trans retinoic acid (ATRA) and arsenic trioxide ex vivo . Compared with 56% penetrance in Ctsg-PML-RARA mice, female homozygous and male hemizygous Kdm6a KO x Vav1 - Cre x Ctsg-PML-RARA mice developed APL with 100% penetrance. We fully characterized pre-leukemic hematopoiesis in young (6 to 8-weeks-old) Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (compared to appropriate littermate controls) to define the underlying mechanism(s) by which Kdm6a inactivation contributes to leukemogenesis. Like our double cross Kdm6a conditional KO x Vav1 - Cre mice, the loss of Kdm6a expression in young female homozygous triple cross mice caused mild thrombocytopenia and splenomegaly. These young mice had neutrophilia and lymphopenia, which the female Kdm6a -null double cross mice only acquired with age. We observed a trend for increased numbers of colonies in a myeloid serial replating assay that was also specific to this cohort. Next, we demonstrated that this cohort had significant decreases in the Kit+Lin-Sca-1+ (KLS) cell, short-term hematopoietic stem cell (ST-HSC), and multipotent progenitor (MPP) hematopoietic stem and precursor compartments. This recapitulated the findings from the young, double cross Kdm6a -null female mice but was more pronounced. We found a concomitant increase in granulocyte-macrophage progenitors (GMPs) and decrease in megakaryocyte-erythroid progenitors (MEPs) that was also specific for the female Kdm6a -null triple cross cohort. Finally, we performed gene expression profiling via exon arrays on flow-sorted SLAM cells from aged (1-year-old) female Kdm6a -null double cross mice and aged controls. Unsupervised hierarchical clustering revealed a clear distinction between cohorts. We identified 1520 differentially expressed genes by an ANOVA analysis (fold change > 2, P < 0.05) between the Kdm6a -null versus WT aged SLAM cells. The list of differentially expressed genes included Pml (downregulated in Kdm6a -null SLAM cells with fold change of 2.35) and Flt3 (upregulated in Kdm6a -null SLAM cells with fold change of 2.32). Expression changes of these two genes may be associated with the mechanism of how inactivation of Kdm6a cooperates with PML-RARA in our mouse model. Pml haploinsuffiency has been show to modestly cooperate with PML-RARA in mouse models of APL, and FLT3 mutations are the most common secondary mutations in APL. In an effort to further elucidate the mechanism by which loss of Kdm6a contributes to malignant hematopoiesis, we have completed RNA-Seq, ATAC-Seq, anti-histone H3 (monomethyl K4) ChIP-Seq, and anti-histone H3 (trimethyl K27) ChIP-Seq studies on mouse APL samples derived from Ctsg-PML-RARA mice that developed spontaneous leukemias (n = 3 Kdm6a -WT samples versus n = 3 Kdm6a -mutant samples). Analysis of this data set is now underway. In sum, our data have demonstrated a functional role of Kdm6a in malignant hematopoiesis and have improved our understanding of its subsequent connection to leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Whole Genome Sequencing of Spontaneously Occurring Rat Natural Killer Large Granular Lymphocyte Leukemia Identifies JAK1 Somatic Activating Mutation.

Large granular lymphocyte (LGL) leukemia arises spontaneously in elderly Fischer (F344) rats. This rodent model has been shown to emulate many aspects of the natural killer (NK) variant of human LGL leukemia. Previous transplantation of leukemic material into young F344 rats resulted in several strains of rat NK (RNK) primary leukemic cells. One strain, RNK-16, was adapted into the RNK-16 cell line and established as an aggressive NK-LGL leukemia model. Whole genome sequencing of the RNK-16 cell line identified 255,838 locations where the RNK16 had an alternate allele that was different from F344, including a mutation in Jak1. Functional studies showed Jak1 Y1034C to be a somatic activating mutation that mediated increased STAT signaling, as assessed by phosphoprotein levels. Sanger sequencing of Jak1 in RNK-1, -3, -7, and -16 found only RNK-16 to harbor the Y1034C Jak1 mutation. In vivo studies revealed that rats engrafted with RNK-16 primary material developed leukemia more rapidly than those engrafted with RNK-1, -3, and -7. Additionally, ex vivo RNK-16 spleen cells from leukemic rats exhibited increased STAT1, STAT3, and STAT5 phosphorylation compared to other RNK strains. Therefore, we report and characterize a novel gain-of-function Jak1 mutation in a spontaneous LGL leukemia model that results in increased downstream STAT signaling.

Congenital neutropenia in the era of genomics: classification, diagnosis, and natural history.

This review focuses on the classification, diagnosis and natural history of congenital neutropenia (CN). CN encompasses a number of genetic disorders with chronic neutropenia and, for some, affecting other organ systems, such as the pancreas, central nervous system, heart, bone and skin. To date, 24 distinct genes have been associated with CN. The number of genes involved makes gene screening difficult. This can be solved by next-generation sequencing (NGS) of targeted gene panels. One of the major complications of CN is spontaneous leukaemia, which is preceded by clonal somatic evolution, and can be screened by a targeted NGS panel focused on somatic events.

Time Sequential Transcriptome Analysis Identifies Mir-126 As an Early Biomarker for Inv(16) Acute Myeloid Leukemia (AML) Disease Progression

Inv(16)(p13q22)/t(16;16)(p13.1;q22) is one of the most common chromosomal mutations found in human acute myeloid leukemia (AML). This inversion on chromosome 16 creates a fusion gene CBFb-MYH11 and leads to the expression of a fusion protein CBFβ-SMMHC. Expression of Cbfβ-SMMHC (CM) in a conditional Cbfb-MYH11 knock-in mouse model (Cbfb+/56M/Mx1-Cre) leads to development of spontaneous AML with the acquisition of additional genetic and epigenetic alterations. In order to further understand the underlying mechanism(s) driving leukemogenesis and identify predictive biomarkers during disease progression, we performed RNA-seq to track changes of transcriptiome in time series peripheral blood samples using illumina HiSeq sequencer. A cohort of 16 mice were included in the experiment, 9 out of which are CM conditional knock-in mice and the remaining 7 are controls. Peripheral blood samples were collected before the induction of CM expression as the "0" time point samples. After induction, peripheral blood samples were collected at 1, 2, 3.5, 4.5, 5.5 and 6.5 month (experimental end point) after induction, and leukemic samples were collected when mice were moribund as the "end" time point samples. Between 3.5 and 6.5 months, 6 out of 9 CM knock-in mice in this cohort had succumbed to lethal leukemia. Total RNA was isolated for mRNA and microRNA (miR) sequencing library preparation. The mapped sequencing read counts were annotated to genes and differential expression was compared using edgeR. False discovery rate (FDR) were used to adjust for multiple comparisons. Genes with adjusted P value (FDR) <0.05 and fold-change >2 were considered differentially expressed genes. Unsupervised clustering analysis revealed that all diseased mice showed unique disease-related mRNA and miRNA signatures at the end point. The disease-related signatures were absent in all control mice and the three CM conditional knock-in mice that did not develop leukemia. We found that 2,032 mRNA genes and 106 miRs were significantly up-regulated whereas 2,926 mRNA genes and 121 miRs were significantly down-regulated at the leukemia end point. Principal component analysis revealed that the disease-related changes occur quite early, at 1-2 month after induction. Among the earliest changes, miR-126-3p and miR-126-5p, previously reported to be associated with human inv(16) AML, were significantly up-regulated (log fold change =2.09 and 2.36 respectively; p< 0.0001) at 1 month and increased progressively throughout leukemia progression (log fold change = 4.11 and 4.45; p< 0.0001). DAVID gene ontology (GO) analysis of genes negatively correlated with miR-126 expression showed significant enrichment for mitotic cell cycle and cell division GO pathways. In addition, dynamic changes in a number of predicted miR-126 targets were seen during disease progression. We reasoned that high miR-126 expression might contribute to AML progression. Lentiviral miR-126 knock-down in AML LSK (Lin-/Sca1+/cKit+) cells from CM induced mice led to increased apoptosis and reduced quiescence in vitro. To assess the role of miR-126 in leukemia progression in vivo, we then designed a novel CpG-miR-126 inhibitor (Zhang et al, submitted). Mice transplanted with CM+ AML and treated with with anti-miR126 (5 mg/kg, daily, 30 days) had a significant delay in leukemia progression and enhanced survival (control 42% survival v.s. anti-miR-126 80% survival at 70 days; n=7-10), supporting the notion that high miR-126 contributes to disease growth. Further mechanistic investigation of miR-126 function during inv(16) AML progression are ongoing. In conclusion, transcriptome analysis of time series samples during spontaneous leukemia progression allows identification of disease-related changes and early increase of miR-126 as a potential biomarker. Further functional validation is expected to provide mechanistic insights and rational for targeting miR-126 in inv(16) AML. DisclosuresStein:Seattle Genetics: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Stemline Therapeutics: Consultancy, Research Funding; Argios: Research Funding; Celgene: Research Funding.

Abstract 3729: Novel radiation mitigators and anticancer drugs

Abstract The possibility of a radiation disaster from a nuclear detonation or accident has existed for over 50 years and spawned much of the basic research in radiobiology in the 1950-60s. The recent Fukushima accident was yet another reminder that there remains a dire need to develop novel therapies against radiation-induced toxicities. Here we report on the development of two novel radiation countermeasure therapies: CJ010 and Yel002. These small, biologically active, drug-like molecules were uncovered in the DEL high throughput assay reducing radiation-induced cyto- and geno-toxicity in yeast. Radiation-modulating activity was further confirmed in yeast plate-based DEL Assay: addition of either CJ010 or Yel002 to irradiated cultures reduced cell death and genomic instability. Further, the compounds increases survival to 75% in vivo following an LD100/30 dose of ionizing radiation (IR) with the first therapeutic injection administered 24 hours post exposure followed by injections at 48,72,96, and 120 hours. Additionally, treatment with Yel001 an analog of CJ010 and Yel002 compounds reduces radiation-induced leukemia from 90% to to 50% and 40% respectively. Of note, treatment with either Yel001 or Yel002 reduced spontaneous leukemia rate from 10% to 0%. Treatment with Yel002 following IR accelerates the recovery of the hematopoietic cells after sub-lethal exposures. In addition, treatment with Yel002 reduces EMS, MMS, UV, radioactive iodine, cigarette smoke extract as well as nitrogen mustard induced toxicity as well as genotoxicity showing a broad application spectrum. It also prolongs live of cells in a senescence assay. In addition Atm deficient mice live 16 weeks longer with weekly injection of Yel002 which is about 12 years in human life expectancy. In addition, Yel002 complements a zebrafish model of Diamond Blackfan Anemia. It works in yeast, CHO cells, different human cells, mice and zebrafish. Toxicity has not been observed in neither in vitro or in vivo administrations. Overall, Yel compounds have much potential as stockpile therapies for radiation-induced lethality and cancer: they are highly effective when administered up to 24hours post exposure, they reduce radiation-induce sequelae such as leukemia, and appear to have an acceptable toxicity profile. Citation Format: Robert H. Schiestl, Yelena Rivina, Michael Davoren. Novel radiation mitigators and anticancer drugs. [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 3729.

Multifunctional Fanconi proteins, inflammation and the Fanconi phenotype

Multifunctional Fanconi proteins, inflammation and the Fanconi phenotype

Functional and Molecular Consequences of the Dnmt3aR882H Mutation in Acute Myeloid Leukaemia

The de novo DNA Methyltransferase 3A (DNMT3A) gene is one of the most commonly mutated gene in acute myeloid leukaemia with a normal karyotype (AML-NK). Approximately two thirds of mutations are heterozygous and affect codon R882, located within the methytransferase domain. Also, we and others have shown that these mutations occur in a significant proportion of haematologically normal people and behave as leukaemia-initiating changes.DNMT3A mediates the transfer of a methyl group from s-adenosylmethionine (SAM) to the 5' carbon of cytosine residues at CpG dinucleotides. The DNMT3A protein domains bind to unmethylated H3K4, a repressive histone mark, as well as the H3K36Me3 mark associated with active transcription. DNA methylation is linked to histone modifications and DNMT3A binds to class I and II histone deacetylases (HDAC) and histone methyltransferases (HMT), such as SETDB1 and G9a, as well components of Polycomb repressive complex 2 (PRC2).DNMT3AR882H has been reported to exert a dominant-negative effect on wild type (WT) DNMT3A, with a significant reduction of its enzymatic activity. In AML samples, the R882 mutation correlates with global hypomethylation, particularly at CpG islands, shores and promoters, although some promoter hypermethylation has also been reported. However, the molecular mechanism, through which DNMT3AR882H alters methylation to drives leukaemia, remains unknown.In order to understand the molecular and functional consequences of the DNMT3AR882H mutation in leukaemogenesis we developed a mouse model (Dnmt3afloxR882H/+), whereby targeting of the Dnmt3a allele does not inactivate the native locus. This allows conditional expression of the mutant protein within haematopoietic stem and progenitor cells that have developed in the presence of normal levels of Dnmt3a. Dnmt3afloxR882H/+ mice were crossed with mice carrying an interferon-inducible Cre recombinase allele (Mx1-Cre). Upon Cre induction with pIpC the endogenous Dnmt3a exon 23 (last exon), was efficiently replaced by the human exon 23 carrying the R882H mutation. This was confirmed at both the DNA and the RNA level. In order to investigate the molecular effects of Dnmt3aR882H, we analysed pre-leukaemic haematopoietic cells (in mice 5-6 weeks post Cre-induction). We observed that Dnmt3aR882H/+ mutant bone marrow (BM) cells had markedly enhanced self-renewal potential as manifested by increased serial replating in cytokine supplemented methylcellulose media in comparison to WT mice. We next examined the relative haemopoietic reconstitution efficiency of Dnmt3aR882H/+ BM cells in a competitive setting. Dnmt3aR882H/+ BM (CD45.2) cells were mixed with WT competitor BM (CD45.1) cells at a ratio of 1:2 and transplanted into lethally irradiated recipient mice (CD45.1/45.2). The analysis of blood chimerism in recipients indicated an increased contribution from Dnmt3aR882H/+ in comparison to WT cells, indicating the enhanced ability of Dnmt3aR882H/+ BM cells to engraft and reconstitute recipient mice. Currently, we are addressing the role of Dnmt3aR882H in the spontaneous leukaemia development alone as well as in rational combinations with other mutations present in human AML.Additionally, to elucidate the molecular mechanism underlying the striking haematopoietic phenotypes ofDnmt3aR882H/+ we have performed analyses of DNA methylation and hydroxymethylation patterns in the haematopoietic progenitor compartment (lineage negative cell population) of Dnmt3aR882H/+ and WT mice, using reduced representation oxidative bisulfite sequencing (RRoxBS) in the pre-leukaemic setting. Moreover, due to cross-talk between Dnmt3a mediated DNA methylation and other chromatin states, we have analysed Dnmt3aR882H-associated changes in histone tail modifications, using ChIP-Seq. As the DNA and histone modifications associated with Dnmt3aR882H are likely to lead to alter global gene expression, we also performed RNAseq analysis in Dnmt3aR882H/+ and WT mice.Currently, we are conducting an integrated analysis, coupling DNA and histone modifications to aberrant gene expression and Dnmt3aR882H-dependent phenotypes. We predict that this work will provide a detailed understanding of the molecular and cellular effects of DNMT3A-R882H and in so doing reveal rational therapeutic approaches for patients with this common and aggressive subtype of AML. DisclosuresNo relevant conflicts of interest to declare.

Genetic Inhibition of PI3K p110delta Antagonizes Survival Signals and Induces Immune Activation in Chronic Lymphocytic Leukemia (CLL)

Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) p110δ pathway. In addition, immune tolerance, a critical mechanism for cancer immune invasion, remains a major barrier for effective anti-cancer therapy. PI3K p110δ also plays critical roles in T cell development and function. Studies using genetic p110δ kinase-dead mice have showed that p110δ inhibition can reverse the tumor induced immune tolerance, impairs the expansion of regulatory T cells (Treg) thereby enhancing the cytotoxic CD8+ T cells to induce tumor regression in various solid tumor models.Methods: Here we use a PI3K p110δ kinase-dead (δD910A/D910A) mouse model and a well-characterized Eμ-TCL1 mouse CLL model to delineate the role of p110δ signaling in CLL leukemia pathogenesis and microenvironmental immune tolerance. The δD910A/D910A has a point mutation on the p110δ kinase domain, which results in the complete inactivation of p110δ activity without affecting the expression of p110δ or the other isoforms. TCL1 mice exhibit B-cell leukemia similar to that in humans beginning around 4-6 months. Adoptive transfer experiments are performed by tail-vein injection of 5e6 splenic lymphocytes from a leukemic Eμ-TCL1 mouse. Leukemia progression is measure by flow cytometry for the CD5+ CD19+ % lymphocytes in the peripheral blood. Whole blood cytotoxicity assay was performed by incubating whole blood from recipient mice with cryopreserved cells from the original donor to assess the their cytotoxic effects towards the donor cells.Results: δD910A/D910A mice were crossed with TCL1 mice to examine the effect of systemic p110δ inactivation in CLL. We show that global inactivation of PI3K p110δ significantly prevented spontaneous leukemia development. Both δWT /TCL1 and δWT/D910A /TCL1 mice developed CLL like disorder starting 4 months whereas δD910A/D910A /TCL1 mice did not show any sign of disease up to 14 months. This indicates that p110δ is a critical kinase for CLL disease initiation and expansion.We then performed adoptive transfer of TCL1 leukemic cells into p110δ mice to examine the role of p110δ in the microenvironment. We show that inhibiting p110δ in the microenvironment impairs leukemia engraftment. On day 31 after engraftment, 7 out of 10 mice from the δWT group developed leukemia while no mice from δD910A/D910A group had detectable leukemia. On day 43, δD910A/D910A group started to have leukemia incidence (2 out of 7) when δWT group continued to progress. δWT/D910A group showed intermediate leukemia incidence and disease development, which were different from the phenotype we saw in δWT/D910A /TCL1. This suggests that p110δ plays a different role outside of the leukemia compartment.To identify potential effector cells, we performed adoptive transfer of ovalbumin (OVA) -expressing TCL1 leukemia cells into p110δ mice. The TCR (OT1) specific SIINFEKL peptide of OVA is used to induce the expansion of OVA antigen specific CD8+ T cells. Interestingly, upon engraftment, δWT and δWT/D910A mice showed similar levels (4% and 3.5%, respectively) of OVA specific CD8+ T cells whereas the δD910A/D910A mice only have 2%. This appears to be contradicting to the less leukemia development we observed previously. However, in vitro whole blood killing assay showed that whole blood from δD910A/D910A mice exhibited higher cytotoxicity to TCL1 leukemia cells despite their impaired development of the antigen specific anti-tumor CD8+ T cells.Conclusions: This study demonstrates that blocking p110δ in CLL not only abrogates survival signals in leukemic cells but also has the potential to reverse immune suppression in the micoenvironment. Studies are ongoing to pinpoint the effector cell subsets. The finding from this study will suggest that idelalisib, the PI3K p110δ inhibitor recently approved by FDA for the treatment of CLL, or other inhibitors of this pathway can potentially be used to treat a wide range of cancers by unleashing host anti-tumor immunity. DisclosuresByrd:Acerta Pharma BV: Research Funding.

Abstract CN10-03: Childhood obesity and leukemia: Opportunities for intervention

Abstract Obesity is a major contributor to cancer mortality, and is responsible for ~90,000 cancer deaths per year in the U.S. Part of this association can be attributed to the effect of obesity to increase cancer incidence, while obesity has been shown to worsen the outcome from a number of types of cancer. Retrospective studies have shown that obese children diagnosed with the high-risk form of the most common childhood cancer, acute lymphoblastic leukemia (HR-ALL) have a substantially higher risk of relapse than lean children. We have investigated these associations, generating laboratory models to investigate how obesity might impact ALL progression and treatment. Using two mouse models, we found that obesity accelerates the appearance of spontaneous leukemia. We have further discovered that obese mice implanted with leukemia had poorer survival after treatment with either vincristine or L-asparaginase. We have identified several effects of adipocytes which appear to contribute to this worse outcome. Adipocytes attract ALL cells to migrate into adipose tissue via secretion of the chemokine CXCL12, protect ALL cells from a number of chemotherapies, absorb chemotherapy leading to unfavorable pharmacokinetics, and secrete glutamine and possibly other fuels to support leukemia cell proliferation. In all, adipocytes have a number of effects which likely contribute to the worse prognosis in obese patients with ALL. These effects are particularly concerning given the high prevalence of overweight and obesity in leukemia patients. Two-thirds of adults and one-third of children in the U.S. are either overweight or obese. Almost half of new patients with HR-ALL in our institution are overweight and obese. Furthermore, we have observed that the body fat of these patients increases by ~20% during the first month of treatment, and even further by several months into therapy. HR-ALL patients therefore have a large and increasing burden of adipose tissue which may directly impair leukemia treatment and worsen prognosis. We now have evidence that the effect of obesity might be reversible. A retrospective analysis of HR-ALL patients showed that those who were obese at diagnosis, but lost weight and became non-obese for more than half of their treatment, had an improved outcome, similar to patients who were never overweight or obese. Experiments in our laboratory demonstrated that switching obese mice to a low-fat diet at start of ALL treatment substantially improves outcome from vincristine treatment, compared to those continued on the high fat-diet (92% vs. 17% survival, p&amp;lt;0.001). In fact, this dietary intervention led to a better survival than even control mice raised on the same low-fat diet. These bench and clinical findings set the stage for an obesity-targeted intervention in children with ALL. We hypothesize that targeting obesity during leukemia treatment will reduce body fat gain, improve quality of life, and potentially improve outcome. Citation Format: Steven D. Mittelman. Childhood obesity and leukemia: Opportunities for intervention. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr CN10-03.

Abstract 1793: Novel radiation mitigators and anticancer drugs

Abstract The possibility of a radiation disaster from a nuclear detonation or accident has existed for over 50 years and spawned much of the basic research in radiobiology in the 1950-60s. The recent Fukushima accident was yet another reminder that there remains a dire need to develop novel therapies against radiation-induced toxicities. Here we report on the development of two novel radiation countermeasure therapies: Yel001 and Yel002. These small, biologically active, drug-like molecules were uncovered in the DEL high throughput assay reducing radiation-induced cyto- and geno-toxicity in yeast. Radiation-modulating activity was further confirmed in yeast plate-based DEL Assay: addition of either Yel001 or Yel002 to irradiated cultures reduced cell death and genomic instability. Further, Yel compounds increases survival to 75% in vivo following an LD100/30 dose of ionizing radiation (IR) with the first therapeutic injection administered 24 hours post exposure followed by injections at 48,72,96, and 120 hours. Additionally, treatment with Yel001 and Yel002 compounds reduces radiation-induced leukemia from 90% to to 50% and 40% respectively. Of note, treatment with either Yel001 or Yel002 reduced spontaneous leukemia rate from 10% to 0%. Furthermore, Yel002 significantly prolonged the life of Atm deficient mice. Treatment with Yel002 following IR accelerates the recovery of the hematopoietic cells and protects stem cells after sub-lethal exposures. In addition, treatment with Yel002 reduces I131, EMS, MMS, UV, cigarette smoke extract as well as nitrogen mustard induced toxicity as well as genotoxicity showing a broad application spectrum. Proteomics shows that Yel002 induces DNA repair mediated by Atm signaling, homologous recombination, nonhomologous end joining, base excision repair, DNA damage binding proteins and chromosome segregation functions. Toxicity has not been observed in neither in vitro or in vivo administrations, even in a two generation teratogenicity assay. Overall, Yel compounds have much potential as stockpile therapies for radiation-induced lethality and cancer as well as spontaneous cancer: they are highly effective when administered up to 24hours post exposure, they reduce radiation-induced sequelae such as leukemia, and appear to have an acceptable toxicity profile. Citation Format: Robert H. Schiestl, Michael Davoren, Yelena Rivina. Novel radiation mitigators and anticancer drugs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1793. doi:10.1158/1538-7445.AM2015-1793

Abstract B44: Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton's tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. The PI3K p110δ inhibitor, idelalisib, was granted a breakthrough therapy designation by the FDA in relapsed CLL and the BTK inhibitor, ibrutinib, has received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies. Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, IPI-145, which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of IPI-145 in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM IPI-145 for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to IPI-145, displaying about 20% decrease in viability at concentrations greater than 0.5μM, however the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM IPI-145 for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, IPI-145 is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, IPI-145. This study provides solid rationale for ongoing clinical development of IPI-145 in CLL patients. Additionally, as IPI-145 can continue to inhibit the PI3K/AKT pathway in the setting of a C481S BTK mutation, it provides justification for clinical studies of this agent in this subset of patients. Citation Format: Amy J. Johnson, Shuai Dong, Daphne Guinn, Jason A. Dubovsky, Yiming Zhong, Amy Lehman, Jeff Kutok, Jennifer A. Woyach, John C. Byrd. Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B44.

Pharmacologic Inhibition with Duvelisib (IPI-145) and Genetic Inhibition of PI3K p110δ Antagonizes Intrinsic and Extrinsic Survival Signals in Chronic Lymphocytic Leukemia (CLL)

Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen, and bone marrow. CLL cells display up-regulated B-cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton’s tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. Both the PI3K p110δ inhibitor, idelalisib, and the BTK inhibitor, ibrutinib, have received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies.Methods/Results: Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, duvelisib (IPI-145), which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of duvelisib in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM duvelisib for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to duvelisib, displaying about 20% decrease in viability at concentrations greater than 0.5μM; however, the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM duvelisib for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, duvelisib is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in the leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose-dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, duvelisib. DisclosuresDubovsky:Principia Inc.: Research Funding. Kutok:Infinity Pharmaceuticals, Inc.: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

The Effect of Synthetic and Natural Antioxidants on the Emergence and Development of Malignant Tumors

The effect of different doses of synthetic antioxidant b-(4-hydroxy- 3,5-ditertbutylphenyl), propionic acid (phenosan) and savory essential oil on the development of spontaneous leukemia was studied. The drug efficiency was determined from the survival curves, animal life spans, and the incidence of leukemia. Phenosan is characterized by clear antitumor activity in therapeutic (10 -4 mol/kg) and ultra-low (10 -14 mol/kg) doses. The dose of 10 -14 mol/kg increases the life span. The savory essential oil in low doses mixed with drinking water (150 ng/ml) or food (2,5 mg/g) increased the average lifetime of mice by 20-35 %. The low doses of these drugs seem promising as preventive drugs.

Abstract 4909: Novel radiation mitigators and cancer chemoprevention drugs

Abstract The possibility of a radiation disaster from a nuclear detonation or accident has existed for over 50 years and spawned much of the basic research in radiobiology in the 1950-60s. The recent Fukushima accident was yet another reminder that there remains a dire need to develop novel therapies against radiation-induced toxicities. Here we report on the development of two novel radiation countermeasure therapies: Yel001 and Yel002. These small, biologically active, drug-like molecules were uncovered in the DEL high throughput assay reducing radiation-induced cyto- and geno-toxicity in yeast. Radiation-modulating activity was further confirmed in yeast plate-based DEL Assay: addition of either Yel001 or Yel002 to irradiated cultures reduced cell death and genomic instability. Further, Yel compounds increases survival to 75% in vivo following an LD100/30 dose of ionizing radiation (IR) with the first therapeutic injection administered 24 hours post exposure followed by injections at 48,72,96, and 120 hours. Additionally, treatment with Yel001 and Yel002 compounds reduces radiation-induced leukemia from 90% to to 50% and 40% respectively. Of note, treatment with either Yel001 or Yel002 reduced spontaneous leukemia rate from 10% to 0%. Furthermore, Yel002 significantly prolonged the life of Atm deficient mice. Treatment with Yel002 following IR accelerates the recovery of the hematopoietic cells and protects stem cells after sub-lethal exposures. In addition, treatment with Yel002 reduces I131, EMS, MMS, UV, cigarette smoke extract as well as nitrogen mustard induced toxicity as well as genotoxicity showing a broad application spectrum. Proteomics shows that Yel002 induces DNA repair mediated by Atm signaling, homologous recombination, nonhomologous end joining, base excision repair, DNA damage binding proteins and chromosome segregation functions. Toxicity has not been observed in neither in vitro or in vivo administrations, even in a two generation teratogenicity assay. Overall, Yel compounds have much potential as stockpile therapies for radiation-induced lethality and cancer as well as spontaneous cancer: they are highly effective when administered up to 24hours post exposure, they reduce radiation-induced sequelae such as leukemia, and appear to have an acceptable toxicity profile. Citation Format: Robert H. Schiestl, Yelena Rivina, Michael Davoren. Novel radiation mitigators and cancer chemoprevention drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4909. doi:10.1158/1538-7445.AM2014-4909

Leukemogenesis in heterozygous PU.1 knockout mice.

Most murine radiation-induced acute myeloid leukemias involve biallelic inactivation of the PU.1 gene, with one allele being lost through a radiation-induced chromosomal deletion and the other allele affected by a recurrent point mutation in codon 235 that is likely to be spontaneous. The short latencies of acute myeloid leukemias occurring in nonirradiated mice engineered with PU.1 conditional knockout or knockdown alleles suggest that once both copies of PU.1 have been lost any other steps involved in leukemogenesis occur rapidly. Yet, spontaneous acute myeloid leukemias have not been reported in mice heterozygous for a PU.1 knockout allele, an observation that conflicts with the understanding that the PU.1 codon 235 mutation is spontaneous. Here we describe experiments that show that the lack of spontaneous leukemia in PU.1 heterozygous knockout mice is not due to insufficient monitoring times or mouse numbers or the genetic background of the knockout mice. The results reveal that spontaneous leukemias that develop in mice of the mixed 129S2/SvPas and C57BL/6 background of knockout mice arise by a pathway that does not involve biallelic PU.1 mutation. In addition, the latency of radiation-induced leukemia in PU.1 heterozygous mice on a genetic background susceptible to radiation-induced leukemia indicates that the codon 235 mutation is not a rate-limiting step in radiation leukemogenesis driven by PU.1 loss.

Lymphoid metastasis of rat My2/De leukemia

By grafting spontaneous leukemia tumor cells, the myeloid My2/De leukemia rat model was established. Death was caused by impaired functions of heavily infiltrated organs. In vitro culturing of tumor cells, blood and bone marrow counts and cytochemic reactions indicated the leukemic the origin resembling human myeoloblastic leukemia. Metastatic spread was followed after i.v. and i.p. injection, and by implantation of leukemia cells under the renal capsule of rats. Primary tumor and metastasis formation was visualized by (18)FDG or (11)C-methionine administration and MiniPET. The accumulation of radiotracers was measured in different organs and expressed as Differential Absorption Ratios (DARs). Subrenal implantation of My2/De cells resulted in their appearance in other abdominal organs and in parathymic lymph nodes. The release of tumor cells from the primary kidney to the peritoneum was mimicked by the i.p. administration of ink particles. Ink particles deposited in the abdominal organs and in the thoracal lymph nodes, preferentially in parathymic lymph nodes, confirming the notion of lymphatic spread of metastasis.

Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL)

AbstractChronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton's tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

The CD37-targeted antibody–drug conjugate IMGN529 is highly active against human CLL and in a novel CD37 transgenic murine leukemia model

Therapeutic regimens for chronic lymphocytic leukemia (CLL) have increasingly utilized monoclonal antibodies since the chimeric anti-CD20 antibody rituximab was introduced. Despite improved clinical outcomes, current CLL therapies are not curative. Therefore, antibodies with greater efficacy and novel targets are desirable. One promising target is CD37, a tetraspanin protein highly expressed on malignant B-cells in CLL and non-Hodgkin lymphoma. While several novel CD37-directed therapeutics are emerging, detailed preclinical evaluation of these agents is limited by lack of appropriate animal models with spontaneous leukemia expressing the human CD37 (hCD37) target. To address this, we generated a murine CLL model that develops transplantable hCD37+ leukemia. Subsequently, we engrafted healthy mice with this leukemia to evaluate IMGN529, a novel hCD37-targeting antibody-drug conjugate. IMGN529 rapidly eliminated peripheral blood leukemia and improved overall survival. In contrast, the antibody component of IMGN529 could not alter disease course despite exhibiting substantial in vitro cytotoxicity. Furthermore, IMGN529 is directly cytotoxic to human CLL in vitro, depletes B-cells in patient whole blood, and promotes killing by macrophages and NK cells. Our results demonstrate the utility of a novel mouse model for evaluating anti-human CD37 therapeutics and highlight the potential of IMGN529 for treatment of CLL and other CD37-positive B-cell malignancies.

All-Trans Retinoic Acid Synergizes With FLT3 Tyrosine Kinase Inhibition To Eliminate FLT3/ITD-Expressing Leukemia Cells

FLT3 is one of the most frequently mutated genes in AML with approximately 1/3 of patients affected, and the internal tandem duplication (ITD) mutations portending a poor prognosis. To improve the cure rate for FLT3 mutant AML a number of FLT3 tyrosine kinase inhibitors (TKIs) have been developed to inhibit FLT3 signaling. While several recent FLT3 TKIs are proving increasingly successful at achieving high levels of FLT3 inhibition, there remain several problems in treating FLT3 mutant AML. One is that these drugs used as monotherapy achieve limited clinical responses and do not cure patients. While there is hope that combination with chemotherapy will achieve an improved cure rate in these patients without the necessity for hematopoietic stem cell transplantion (HSCT), the ultimate goal of studies are to achieve cures without chemotherapy, eliminating the short and long-term side effects that its use engenders. Thus, finding additional molecular targets that might synergize with FLT3 inhibition will move the field towards the goal of eliminating chemotherapy. One pathway known to play important roles in leukemia stem cell (LSC) survival and differentiation is the retinoic acid (RA) pathway. We therefore explored the combination of molecularly targeting the RA pathway together with FLT3 TKIs to determine the effect on FLT3 mutant leukemia cells.FLT3/ITD+ AML cell lines (Molm14 and MV411), along with FLT3/WT cell lines (THP-1, SEMK2 and NB4), were treated with FLT3 TKI alone (AC220 and sorafenib), ATRA alone, FLT3 TKI plus ATRA, or vehicle controls. Proliferative, apoptotic, cell-cycle and differentiation effects on the cells were assessed by MTT, annexin V binding, propidium iodide (PI) staining, CD11b staining and cell count assays. Highly synergistic effects were observed for the combination of ATRA with FLT3 TKIs against FLT3/ITD+ cells, with combination index (CI) values of 0.1-0.6. Colony forming unit (CFU) assays further demonstrated decreased clonogenicity of Molm14 and MV411 cells upon treatment with ATRA and sorafenib. A series of experiments were performed using a genetic model of spontaneous leukemia that we developed in which FLT3/ITD knock-in mice are bred with NUP98- HOXD13 (NHD13) transgenic mice, resulting in AML that is highly penetrant, lethal and transplantable. Cohorts of leukemic mice were generated by transplanting lineage negative (Lin-) BM cells from leukemic FLT3/ITD-NHD13 mice, and treated with either vehicle, sorafenib, ATRA or a combination of both drugs. We determined that treatment with sorafenib plus ATRA greatly decreases the level of engraftment at 2 and 8 weeks, and increases median survival, with some mice even cured of their disease. We also directly assessed the effect of the treatments (FLT3 TKI alone, ATRA alone, FLT3 TKI plus ATRA, or vehicle alone) on the proliferation of LSCs by using the different treatments on Lin- BM cells harvested from sick FLT3/ITD NHD13 mice ex vivo and found that ATRA further increased the anti-proliferative effect of FLT3 TKIs, with additive/synergistic CI values of 0.6-1. Lin- BM cells were also assessed in vitro in CFU assays of differentiation and clonogenicity, and combinatorial effects were observed.The results of these experiments show good synergy for this drug combination in vitro and improved survival/reduced LSC frequency in vivo. We believe these preclinical data are encouraging for the development of a clinical trial of ATRA plus FLT3 TKI in relapsed/refractory FLT3 mutant AML patients. Disclosures:No relevant conflicts of interest to declare.