Hemopoiesis and Immune Cell Perturbations during Venetoclax Plus Azacytidine Treatment in Acute Myeloid Leukemia

Abstract Treatment of acute myeloid leukemia (AML) in elderly is still challenging. Indeed, high-dose chemotherapy followed by hematopoietic stem cell transplantation with myeloablative regimens is not always feasible because patients are often unfit and have several comorbidities; however, they frequently show multiple negative prognostic factors and have a worse overall survival compared to younger adults. Venetoclax, the first-in-class Bcl-2 antagonist and first approved for treatment of chronic lymphocytic leukemia, inhibits the anti-apoptotic functions of Bcl-2 inducing apoptosis and tumor growth arrest. Venetoclax is also used in combination with azacytidine, or decitabine, or low-dose cytarabine for treatment of elderly newly diagnosed AML. However, several mechanisms of resistance have already been described, such as increased expression of other anti-apoptotic proteins by the leukemic clone. In this case series, we investigated hematopoiesis and immune cell perturbations during venetoclax plus azacytidine treatment in elderly AML patients. A total of six AML patients (M/F, 2/4; median age, 71 years old; range, 63-79 years) were retrospectively evaluated at the Hematology and Transplant Center, University Hospital "San Giovanni di Dio e Ruggi d'Aragona", Salerno, Italy. Patients received a diagnosis of AML based on the 2016 World Health Organization (WHO) criteria and chemotherapy with azacytidine 75 mg/m 2 daily for 7 days per cycle and venetoclax 70 mg/daily. Two patients were NPM1 mutated (one of them also had mutated IDH1, VAF 27.2%), while all subjects had FLT3 wild type. Based on the 2017 European LeukemiaNet risk classification, two patients had favorable risk and four intermediate. Median follow-up was 10.1 months (range, 4.9-16.6 months), and all patients were in partial or complete remission at the time of writing. Flow cytometry immunophenotype, complete blood counts (CBCs), and WT1 expression levels were performed at diagnosis and after every cycle of therapy as per our institutional guidelines. In our case series, leukemic cells were already decreased after the first cycle of therapy (blasts by flow cytometry + SD, 54.7+39.9% vs 4.2+5.4%, diagnosis vs post I cycle; P = 0.0671; paired t-test performed), while normal granulocytes detected by flow cytometry recovered only after the third cycle of therapy (20.7+23.7% vs 53+6.6%, diagnosis vs post III cycle; P = 0.1396; uncorrected Fisher's mixed model performed). Treated patients also displayed a contextual decreased in WT1 expression levels (normalized WT1 copy number + SD, 1810+2723 copies vs 201+132.9 copies, diagnosis vs post I cycle; P = 0.2660; paired t-test performed). Platelet count tended to increase after the first cycle (P = 0.0680); however, at the end of the second cycle, half of patients were again thrombocytopenic (platelets < 100 x 10 3/µL). Interestingly, percentage of lymphocytes detected by flow cytometry were significantly increased after the second cycle of azacytidine plus venetoclax compared to baseline and after the first cycle of therapy (mean+SD, 13.5+13.3% vs 48+8.7%, diagnosis vs post II cycle; P = 0.0167; and vs 28+11.3%, vs post III cycle; P = 0.0480), likely because an increase in Natural Killer (NK) cell frequency peaking after the second cycle (mean+SD, 19.4+4.4% vs 32.5+15.1%, diagnosis vs post II cycle; P = 0.1383). Moreover, five out of six patients displayed expansion of plasma cells detected by flow cytometry in the bone marrow after the first cycle: in particular, one case patient had expansion of aberrant CD45-/dimCD38++CD138++CD56+CD19- plasma cells, while one subject showed only a transient appearance of clonal plasma cells after the second cycle. No differences in bone marrow monocyte frequencies were described during treatment. Our preliminary results added evidence to efficacy and safety of the combination of venetoclax and azacytidine in treatment of elderly AML in a real-world setting. These drugs might synergistically function on hematopoiesis by inducing apoptosis of neoplastic cells while favoring differentiation of other lineages, as suggested by the expansion of plasma cells, or triggering NK-mediated immunosurveillance. However, prognostic and clinical significance of plasma cell and NK cell expansion in the setting of AML treatment needs to be further explored in larger prospective cohorts. Disclosures No relevant conflicts of interest to declare.

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The Growth Inhibition and Apoptosis Induction of Acute Myeloid Leukemia Blast Population by the Blocking IGF-IR Pathway.

Blood ◽

10.1182/blood.v108.11.4515.4515 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4515-4515

Author(s):

Si Sun ◽

Yanli He ◽

Xingbing Wang ◽

Wei Liu ◽

Jun Liu ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Pi3k Pathway ◽

Leukemia Cell Line ◽

Free System ◽

Acute Myeloid ◽

Marrow Cells

Abstract The insulin-like growth factor-1receptor (IGF-1R) is overexpressed in a variety of tumors and has been associated with cancer development. Here, we analysis the IGF-IR expression on the bone marrow cells from 45 newly diagnosed patients with acute myeloid leukemia (AML) by flow cytometry. IGF-1R universally expressed on AML blasts and the leukemia cell line HL-60, did not show significant correlation with FAB subtypes. However, the bone marrow cells from AML patients with high myeloblast counts (>80%) generally showed brighter IGF-IR expressions, which indicated the IGF-IR pathway might play an important role for AML blast proliferation and survival. Indeed, blocking the IGF-1R pathway by neutralizing monoclonal antibodies could reduce the proliferation of HL-60 cells by 38.28% at 48 hr. This inhibitory effect on blast growth was observed in 4 of 5 AML samples. In the same IGF-1R blocking treatment, the apoptosis of HL-60 cells was significantly induced, resulting in apoptosis of 57% of the cell population with the measurement of Annexin V vs PI staining by flow cytometry. The control contained only 20% apoptotic cells. We also demonstrated that the blockade of the IGF-1R pathway inhibited the phophorylation of the PI3K pathway component Akt in HL-60 cells when cultured in a serum free system with a supplement of 50ng/ml exogenous IGF. Since PI3K pathway activation greatly contributes to the proliferation, survival and drug resistance of AML, it is of interest to study whether blockading IGF-IR could also inhibit the PI3K pathway in primary AML blasts and synergize other anti-leukemia agents to improve the therapeutic effectiveness. Conclusions: IGF-IR may play an important role in the proliferation and survival of the AML blast population; Blocking the IGF-IR pathway could significantly inhibit the growth of AML blasts and considerably induce the apoptosis of AML blasts; IGF-IR could become a critical molecular target in anti-leukemia drug discovery.

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Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.3513.3513 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3513-3513

Author(s):

David B. Sykes ◽

Mark K Haynes ◽

Nicola Tolliday ◽

Anna Waller ◽

Julien M Cobert ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Small Molecules ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Myeloid Differentiation ◽

Prostacyclin Receptor ◽

Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

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The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽

10.1182/blood.v126.23.3090.3090 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3090-3090 ◽

Cited By ~ 1

Author(s):

Folashade Otegbeye ◽

Nathan Mackowski ◽

Evelyn Ojo ◽

Marcos De Lima ◽

David N. Wald

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

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Stromal CYR61 Confers Resistance to Mitoxantrone Via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.2228.2228 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2228-2228

Author(s):

Xin Long ◽

Laszlo Perlaky ◽

Tsz-Kwong Chris Man ◽

Michele S. Redell

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Stromal Cell ◽

Chemotherapy Resistance ◽

Induced Apoptosis ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. Disclosures No relevant conflicts of interest to declare.

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Impact of Minimal Residual Disease and Chimerism Monitoring at Different Timepoints after Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-141064 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-7

Author(s):

Reyes María Martín-Rojas ◽

Gillen Oarbeascoa ◽

Rebeca Bailén ◽

Ignacio Gómez-Centurión ◽

Luis Miguel Juarez ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Cumulative Incidence ◽

Myeloid Leukemia ◽

Residual Disease ◽

Minimal Residual ◽

Risk Of Relapse ◽

Acute Myeloid

¶ Martin-Rojas RM and Oarbeascoa G contributed equally to this work. INTRODUCTION Relapse is the main cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML). The evaluation of minimal residual disease (MRD) could provide a more accurate assessment of the depth of response, and therefore identify patients with higher risk of relapse. AIMS The aim of our study was to analyze the impact of pre-HSCT flow cytometry (FCM) and molecular MRD together with chimerism and MRD in the early post-HSCT period in patients with AML. METHODS We conducted a retrospective study in patients with complete remission AML who underwent a HSCT between 2008 and 2019 in our center. MRD was analyzed by flow cytometry in bone marrow aspirates and by quantitative RT-PCR (NMP1, RUNX1-RUNX1T1, CBFB-MYH11, KMT2A-MLLT3, WT1) in bone marrow aspirates and/or peripheral blood. MRD was determined within the 30 days preceding the HSCT and at day +30 and +90 post-HSCT. Bone marrow and selected CD34+ lineage chimerism was analyzed by STR (AmpFISTR SGM Plus, Thermo Fisher) at days +30 and +90 post-HSCT. This study was approved by our Institutional Ethics Committee. Data were analyzed using IBM SPSS Statistics version 24 and R version 3.5.1. RESULTS A total of 115 patients were analyzed. Pre-HSCT MRD was negative in 58 patients (50.4%) and positive in 57 patients (49.6%). We found no statistically significant differences in the characteristics between the two groups (Table 1). Median follow up was 39 months (IQR 10.4-55.8). 3-year overall survival (OS) for patients with pre-HSCT negative MRD was 72.5% versus 70.3% in patients with positive MRD (p=0.41), with an event free survival (EFS) of 66.9% versus 66.1 (p=0.48) respectively (Figure 1). Median time to the beginning of immunosuppression withdrawal was 82.5 days (IQR 59-93) for patients with negative MRD and 68 days (IQR 55.3-85.3) for patients with positive MRD (p<0.001). The cumulative incidence of grade II-IV acute graft versus host disease (aGVHD) and moderate-severe chronic GVHD did not show statistically significant differences based on the MRD status. Similarly, the cumulative incidence of relapse and the 2-year mortality was not significantly different between the two groups. Patients with negative MRD at day +30 showed a 2-year OS of 83.5% versus 58.1% in patients with positive MRD (p=0.03) and a EFS of 79.9% versus 48.6% (Figure 2). The cumulative incidence of relapse was more elevated in patients with positive MRD (29.8% versus 13.6%) at day +30. Patients with mixed chimerism (MC) at day +30 showed a significantly lower 3-year OS and EFS than patients with complete chimerism (CC). Likewise, the cumulative incidence of relapse was significantly higher in patients with MC, both if detected in bone marrow aspirate and in CD34+ cells. The multivariate analysis revealed that MRD status at day +30 post-HSCT was an independent prognostic factor for EFS (HR 3.74; 95% CI 1.38-10.1; p=0.009). CONCLUSIONS Patients with AML presenting a positive MRD in the early post-HSCT period and those who show a MC at day +30 post-HSCT have lower EFS, with positive MRD at day +30 being an independent prognostic factor for EFS. The evaluation of MRD and chimerism in the early post-HSCT period is useful to identify patients with higher risk of relapse, who may take advantage of preemptive measures. Disclosures Kwon: Gilead, Novartis, Pfizer, Jazz: Consultancy, Honoraria.

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A Phase I Study of Allogeneic Natural Killer Cell Therapy Generated from Cord Blood Hematopoietic Stem and Progenitor Cells in Elderly Acute Myeloid Leukemia Patients

Blood ◽

10.1182/blood.v126.23.1357.1357 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1357-1357 ◽

Cited By ~ 5

Author(s):

Harry Dolstra ◽

Mieke W.H. Roeven ◽

Jan Spanholtz ◽

Basav Hangalapura ◽

Marleen Tordoir ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Therapy ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Residual Disease ◽

Standard Chemotherapy ◽

Donor Chimerism ◽

Elderly Aml

Abstract Introduction Elderly acute myeloid leukemia (AML) patients have a poor prognosis due to high relapse rates following standard therapy. Natural Killer (NK) cell alloreactivity has found to control relapse in AML in the HLA-mismatched haploidentical allogeneic stem cell transplantation (allo-SCT) setting. Moreover, allogeneic NK cell infusions can induce complete remission (CR) inpatients with advanced AML. As a consequence, adoptive NK cell transfer may be a promising treatment for elderly AML patients, who are not eligible for allo-SCT. Most clinical studies exploited NK cell products enriched from leukapheresis of haploidentical donors containing low numbers of T cells that could have contributed to the observed therapeutic effects and potentially induced graft-versus-host disease (GVHD). Therefore, we have developed a GMP-compliant culture system for the generation of large batches of NK cells from umbilical cord blood (UCB)-derived CD34+ progenitor cells, without T cell contamination. Here, we report results of a phase I dose escalation study (Dutch Trial Register nr. NTR2818) to evaluate the feasibility, safety and toxicity of allogeneic UCB-NK cell infusion following an immunosuppressive preparative regimen in elderly AML patients. Secondary endpoints were NK cell lifespan and the effects on minimal residual disease (MRD). Methods Elderly AML patients not eligible for allo-SCT, and in morphologic CR after standard therapy, were given preparative chemotherapy consisting of Cyclophosphamide (Cy;900 mg/m2/day) and Fludarabine (Flu;30 mg/m2/day) on days -6 to -2. At day 0, UCB-NK cells at a dose of 3, 10 or up to 30x106/kg body weight were infused without IL-2 treatment to study if in vivo expansion could be obtained without IL-2 support. Patients were assessed for toxicity and GVHD. Donor chimerism was measured by Q-PCR for discriminating DNA polymorphisms. NK cell expansion and phenotype were analyzed by flow cytometry. MRD was evaluated by flow cytometry and molecular techniques. Results Twelve AML patients (68-76 years) have been included, all in morphologic CR after 2 to 3 standard chemotherapy courses (n=6), or 1 standard chemotherapy course followed by subsequent treatment with hypomethylating agents (azacitidine or decitabine) (n=6). Patients were treated with Cy/Flu and an escalating dose of partially HLA-matched UCB-NK cells. Four patients had good/intermediate risk, 4 poor risk and 4 very poor risk AML. To date, 9 patients received NK cell products containing a median of 74% highly activated CD56+ NK cells, with <1x104/kg CD3+ T cells and <3x105/kg CD19+ B cells. Remaining non-NK cells were CD14+ and/or CD15+ monocytic and myelocytic cells. Follow up did not show GVHD or toxicity attributed to the NK cells. As expected, preparative Cy/Flu induced a neutropenic period of 20 ± 16 days, but no severe infections were seen. A temporary repopulation and persistence of UCB-NK cells could be detected in peripheral blood between days 1 and 8 post-infusion, which was associated with increased IL-15 plasma levels observed in most patients. Interestingly, donor chimerism increased with higher doses of infused UCB-NK cells, and donor chimerism up to 3.5% was found in bone marrow (BM) at day 7/8. Further UCB-NK cell maturation in vivo was observed by acquisition of CD16 and KIRs, while expression of activating receptors was sustained. Of the 9 treated patients so far, 5 (56%) are still in CR after 43, 35, 31, 5 and 4 months, whereas 4 patients relapsed after 5, 6 (2 pts) and 15 months. Despite morphologic CR during azacitidine treatment, residual disease of 6-7% with a leukemia-associated phenotype could be detected by flow cytometry before NK cell infusion in BM of two patients. In both patients MRD was reduced to less than 0.05% at 90 days after UCB-NK cell therapy following Flu/Cy conditioning. Conclusion These results show that GMP-compliant UCB-NK cell products containing up to 30x106 NK cells/kg body weight can be safely infused in non-transplant eligible AML patients following immunosuppressive chemotherapy. After infusion, UCB-NK cells repopulate, mature and migrate to BM without supporting IL-2 infusion. Since we observed reduction in MRD in patients on treatment with hypomethylating agents, this UCB-NK cell therapy may induce or sustain CR in elderly AML patients, and could serve as an alternative consolidation therapy for patients with refractory AML or provide bridge to allo-SCT. Disclosures Spanholtz: Glycostem Therapeutics: Employment. Tordoir:Glycostem Therapeutics: Employment. Bohme:Glycostem Therapeutics: Employment. Kok:Glycostem Therapeutics: Employment.

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Dissecting Signaling Network Responses to PAK4 Allosteric Modulators (PAMs) in Cell Subsets within Primary Human Acute Myeloid Leukemia Samples

Blood ◽

10.1182/blood.v126.23.3686.3686 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3686-3686

Author(s):

Paul Brent Ferrell ◽

William Senapedis ◽

Alexander Cook ◽

Erkan Baloglu ◽

Yosef Landesman ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Leukemia Stem Cells ◽

Allosteric Modulators ◽

Advisory Committees ◽

Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has a poor outcome with limited treatment options in patients with relapsed or resistant disease. Therapy resistance in AML is likely related to the inadequacy of therapy within leukemia cell subsets, including leukemia stem cells (LSCs). The p21-activated kinase (PAK) family of proteins was shown to be overexpressed in cancer cells and to play a key role in proliferation, survival, and maintenance of cellular structure. The series of orally bioavailable PAK4 allosteric modulators (PAM) have previously been shown to have activity in hematological cancer cell lines, including those derived from acute myeloid leukemia (AML) (Senapedis et al. Blood124, 2208-2208). Understanding how therapies target cellular subsets within primary patient samples could aid drug development by revealing any subset specific drug effects. In this project, we studied the effects of p21-activated kinase 4 (PAK4) modulation in AML samples. PAK4 modulation has been shown to have significant effects on many intracellular signaling pathways, including PI3K/AKT, MAPK/ERK and WNT/β-catenin pathways (Senapedis et al. Blood124, 2208-2208). It is unknown whether PAMs will have similar activity in primary leukemia cells. Likewise, it is currently unclear to what extent PAMs will differentially impact primary cell subsets including leukemia stem cells and non-malignant cell subsets that may be critical to recovery of bone marrow functions. We have previously shown that the single cell biology platform of flow cytometry is well-suited for dissecting clinically relevant signaling network mechanisms in primary human AML (Irish et al. Cell, 118(2):217-28). Methods: Flow cytometry was used to dissect the impact of an orally bioavailable PAM in AML cell lines and primary patient tissue. Cell lines chosen for this study included NRAS mutant KG-1 and Kasumi-1, which carry t(8;21) and express the AML1:ETO fusion protein. Primary AML biopsies were acquired from bone marrow or blood prior to any treatment and patients were identified and consented for this study according to a local Institutional Review Board-approved protocol. AML tissue samples were viably cryopreserved and then assayed ex vivo. Established protocols were used for phospho-specific flow cytometry, fluorescent cell barcoding, and data analysis in Cytobank (Irish et al. Cell, 118(2):217-28, Doxie and Irish, Curr Top Microbiol Immunol. 377:1-21). Results: Differential effects of PAK4 inhibition were observed between cell lines and among cell subsets from AML patient bone marrow. In leukemia cell lines and patient samples, p-ERK and p-S6 showed marked inhibition via PAM, though degree of inhibition varied. In AML patient samples, PAMs blocked signaling responses in p-ERK specifically in AML blasts, but spared normal CD45hi mononuclear cells (0.88 vs. 0.29-fold reduction (arcsinh scale) in p-ERK at 10 nM). Within the AML blast population, CD34+ CD38- and CD34+ CD38+ AML subsets showed similar PAM dose response via p-ERK. Conclusions: Single cell analysis effectively distinguishes effects of PAK4 inhibition via a series of allosteric modulators of PAK4 (PAMs) on leukemia and non-leukemia subsets in the same sample. PAM reduced immediate p-ERK and p-S6 levels in primary leukemia and cell lines. Notably, inhibition in various subsets within human AML was successfully measured by phospho-flow cytometry. Signaling changes in p-ERK were minimal within non-leukemic mature CD45+ mononuclear cells found in primary patient biopsies. Analysis of CD34+ CD38- cells indicates that PAMs could have activity within leukemia stem cells, and, at least, effect the AML progenitors. These findings support further investigation into the mechanism of action and treatment potential of PAMs in AML. Disclosures Senapedis: Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Irish:Novartis: Honoraria; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; InCyte: Research Funding. Savona:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Development of Somatic NRAS Mutation Associated with Rapid Transition from Germline GATA2 Mutation Associated Myelodysplastic Syndrome to Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.3616.3616 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3616-3616 ◽

Cited By ~ 1

Author(s):

Yanqin Yang ◽

Yubo Zhang ◽

Jun Zhu ◽

Catherine E. Lai ◽

Jingrong Tang ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Myelodysplastic Syndrome ◽

Myeloid Leukemia ◽

Research Funding ◽

Bone Marrow Aspirate ◽

Nras Mutation ◽

Trisomy 8 ◽

Acute Myeloid

Abstract There is increasing recognition of the role of inherited germline predisposition for myeloid disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The additional somatic genetic events required for development of a malignant phenotype are however poorly understood. A 25 year old woman was referred to the NHLBI hematology branch in March 2014 for a seven year history of pancytopenia. Her medical history included recurrent pneumonias, oral ulcers, severe varicella infection and arthralgias. Prior bone marrow examinations at ages 21 and 23 at outside institutions reported normocellular marrow, tri-lineage hematopoiesis and mild dyspoiesis. Cytogenetics were remarkable for trisomy 8 in 80% (aged 21) or 90% (aged 23) of metaphases. Previously unrecognized lymphedema was noted on examination. Peripheral blood counts showed WBC 2.28 K/ul [normal range: 3.98-10.04], HGB 9.9 g/dL [11.2-15.7], PLT: 67 K/ul [173-369], ALC: 0.36 K/ul [1.18-3.74] and AMC: 0.06 [0.24-0.86]. Peripheral blood flow cytometry demonstrated decreased CD3+ CD4+ (T) cells, CD19+ (B) cells and NK cells. HLA-DR15 negative. Bone marrow examination showed trilineage hematopoiesis, 50-60% cellularity, mild erythroid predominance and mildly increased, mildly atypical megakaryocytes. Blasts less than 5%. Bone marrow flow cytometry revealed severely decreased B-cells and monocytes, absent B-cell precursors, absent dendritic cells, inverted CD4:CD8 ratio, and atypical myeloid maturation pattern. Cytogenetics demonstrated stable trisomy 8 in 90% of metaphases. On the basis of this assessment the diagnosis of MDS was confirmed. Sanger sequencing revealed a GATA2 L375S mutation in the second zinc finger of known pathogenic significance. Four months later she developed increased fatigue and easy bruising with worsening thrombocytopenia (PLT: 10K/ul). Bone marrow was dramatically changed; now markedly hypercellular (90-100%) with diffuse sheets of immature cells consistent with blasts having fine chromatin, distinct or prominent nucleoli, and visible cytoplasm. Blasts were positive for CD33, CD56, CD64, CD123, and CD163; and were negative for CD34, CD14, and myeloperoxidase. Cytogenetics showed a new trisomy 20 in 65% of metaphases, in addition to previously seen trisomy 8 in 100%. A diagnosis of acute monoblastic leukemia (M5a subtype) was made. At both clinic visits bone marrow aspirate was collected on an IRB approved research sample acquisition protocol. Whole exome sequencing of 1ug DNA was performed using Agilent SureSelect v5 Exome enrichment Kits on an Illumina HiSeq 2000 with 100-bp paired-end reads (Macrogen, Rockville, MD). Data was mapped to hg19 (BWA) and processed using an in-house pipeline (Samtools/Picard/GATK/VarScan/Annovar). Mean read depth of target regions was 157 and 149. There was high correlation between both samples with the exception of a NRAS:NM_002524:exon3:c.C181A:p.Q61K mutation (57 of 180 reads) seen only in the later sample. Confirmatory ultra-deep sequencing for NRAS was performed using Illumina TruSight Myeloid Sequencing Panel on an Illumina MiSeq. No evidence of the NRAS Q61K mutation was found in the earlier March MDS bone marrow sample even when sequenced to a depth greater than 1750 reads (see figure). The mutation was confirmed in the August AML sample at a variant allele frequency of 35%. If heterozygous this would reflect a clone size of 70%, consistent with data from both cytogenetics (new trisomy 20 in 65% of metaphases) and the 76% blasts documented by bone marrow aspirate smear differential. We report here the rapid progression to AML in a patient with germline GATA2 MDS associated with development of a new trisomy 20 karyotype and a NRAS Q61K mutation. The NRAS mutation was not detectable after the patient achieved a complete remission following induction chemotherapy further supporting this association. This NRAS mutation has been implicated in the pathogenesis of multiple cancers by constitutive activation of proliferative signaling. GATA2 associated MDS is a high-risk pre-leukemic condition with the potential for rapid evolution to AML. This is the first report of acquired somatic mutations in the RAS/RTK signaling pathway in the context of germline GATA2 insufficiency associated with acute leukemic transformation. Figure 1. Figure 1. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding.

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Establishment of Prognosis Model in Acute Myeloid Leukemia Based on Hypoxia Microenvironment, and Exploration of Hypoxia-Related Mechanisms

Frontiers in Genetics ◽

10.3389/fgene.2021.727392 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jinman Zhong ◽

Hang Wu ◽

Xiaoyin Bu ◽

Weiru Li ◽

Shengchun Cai ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Immune Cell ◽

Enrichment Analysis ◽

Survival Outcomes ◽

Composition Analysis ◽

Pathway Enrichment Analysis ◽

Prognosis Model ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a highly heterogeneous hematologic neoplasm with poor survival outcomes. However, the routine clinical features are not sufficient to accurately predict the prognosis of AML. The expression of hypoxia-related genes was associated with survival outcomes of a variety of hematologic and lymphoid neoplasms. We established an 18-gene signature-based hypoxia-related prognosis model (HPM) and a complex model that consisted of the HPM and clinical risk factors using machine learning methods. Both two models were able to effectively predict the survival of AML patients, which might contribute to improving risk classification. Differentially expressed genes analysis, Gene Ontology (GO) categories, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to reveal the underlying functions and pathways implicated in AML development. To explore hypoxia-related changes in the bone marrow immune microenvironment, we used CIBERSORT to calculate and compare the proportion of 22 immune cells between the two groups with high and low hypoxia-risk scores. Enrichment analysis and immune cell composition analysis indicated that the biological processes and molecular functions of drug metabolism, angiogenesis, and immune cell infiltration of bone marrow play a role in the occurrence and development of AML, which might help us to evaluate several hypoxia-related metabolic and immune targets for AML therapy.

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Clinical and prognostic relevance of CXCL12 expression in acute myeloid leukemia

PeerJ ◽

10.7717/peerj.11820 ◽

2021 ◽

Vol 9 ◽

pp. e11820

Author(s):

Shi-sen Wang ◽

Zi-jun Xu ◽

Ye Jin ◽

Ji-chun Ma ◽

Pei-hui Xia ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Large Scale ◽

Plasma Cells ◽

Immune Cell ◽

White Blood Cells ◽

Cxcl12 Expression ◽

Go Enrichment ◽

The Impact ◽

Acute Myeloid

Background Accumulating studies have been made to understand the association between CXC chemokine ligand-12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) and acute myeloid leukemia (AML). However, large-scale data analysis of potential relationship between CXCL12 and AML remains insufficient. Methods We collected abundant CXCL12 expression data and AML samples from several publicly available datasets. The CIBERSORT algorithm was used to quantify immune cell fractions and the online website of STRING was utilized for gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The statistical analysis and graphical work were mainly performed via the R software. Results CXCL12 expression was extremely down-regulated in AML. Clinically, low CXCL12 expression was correlated with higher white blood cells (WBCs) (P < 0.0001), more blasts in bone marrow (BM) (P < 0.001) and peripheral blood (PB) (P < 0.0001), FLT3-internal tandem duplications (FLT3-ITD) (P = 0.010) and NPM1 mutations (P = 0.015). More importantly, reduced CXCL12 expression predicted worse overall survival (OS) and event-free survival (EFS) in all AML, non-M3-AML, and cytogenetically normal (CN)-AML patients in three independent cohorts. As for immune cell infiltration, high CXCL12 expressed groups tended to harbor more memory B cells and plasma cells infiltration while low CXCL12 expressed groups exhibited more eosinophils infiltration. GO enrichment and KEGG pathways analysis revealed the potential biological progress the gene participating in. Conclusions CXCL12 is significantly down-regulated in AML and low CXCL12 expression is an independent and poor predictor of AML prognosis. CXCL12 expression level correlates with clinical and immune characteristics of AML, which could provide potential assistance for treatment. Prospective studies are needed to further validate the impact of CXCL12 expression before routine clinical application in AML.

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