Protein Nanomedicine Exerts Cytotoxicity toward CD34+

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Protein nanomedicine exerts cytotoxicity towards CD34+ CD38- CD123+ leukemic stem cells Parwathy Chandran, Keechilat Pavithran, Neeraj Sidharthan, Abhilash Sasidharan, Shantikumar V Nair, and Manzoor Koyakutty ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00361 • Publication Date (Web): 27 Oct 2015 Downloaded from http://pubs.acs.org on October 29, 2015

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ACS Biomaterials Science & Engineering

Protein nanomedicine exerts cytotoxicity towards CD34+ CD38- CD123+ leukemic stem cells

Parwathy Chandrana, c, PhD, Keechilat Pavithranb, MD, DM, Neeraj Sidharthanb, MD, DM, Abhilash Sasidharana,c, PhD, Shantikumar Naira*, PhD, Manzoor Koyakuttya*, PhD a

Amrita Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham University, Kochi, Kerala, India

b

Department of Medical Oncology, Amrita Institute of Medical Sciences and Research Centre, Kochi, Kerala, India c

Nanotechnology Innovation Center of Kansas State (NICKS), Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas, USA

ABSTRACT: The efficacy of protein-vorinostat nanomedicine (NV) is demonstrated in leukemic stem cells (LSC) isolated from refractory acute myeloid leukemia (AML) patient samples, where it successfully ablated both ‘CD34+ CD38- CD123+ LSC’ and non-LSC ‘leukemic blast’ compartments, without inducing myelosuppression or hemotoxicity. Besides, NV also exerted excellent synergistic lethality against leukemic bone marrow cells (BMC) at lower concentrations (0.1 µM) in combination with DNA methyltransferase (DNMT) inhibitor, decitabine. Considering the extermination of resilient LSC and synergism with decitabine, NV shows promise for clinical translation in the setting of a more tolerable and effective epigenetic targeted therapy for leukemia. KEYWORDS: cancer stem cells, leukemic stem cells, protein-vorinostat nanomedicine, acute myeloid leukemia, decitabine

1 ACS Paragon Plus Environment


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Persistence of a resilient, residual population of CSC is clinically implicated in the inability of current conventional chemotherapy to eliminate this disease, causing therapeutic failure and relapse.1 Normal stem cells and CSCs share several functional similarities including quiescent cell cycle status, resistance to apoptosis, and self-renewal potential. 2 The existence of CSC was first demonstrated over two decades ago in acute myeloid leukemia (AML) using xenogeneic transplant models, and consequently, human AML leukemic stem cells (LSC) represent the most well characterized CSC population.3-6

Established line of evidences have conferred LSC with CD34+ CD38- CD123+

immunophenotype, owing to preferential overexpression of IL-3 receptor α, CD123 (IL3-Rα) within CD34+ CD38- subset of AML cells, over healthy CD34+ CD38- hematopoietic stem cells (HSC).7-8 Aggressive chemoregimens which mostly target rapidly dividing leukemic blasts often miss their hit over LSCs possessing low mitotic index. Moreover, considering AML as an elderly malignancy with majority of affected population above 60 years of age, intense chemotherapy leads to acute myelosuppression, ensuing infections and related comorbidities, resulting in higher mortality rates (> 40%). Hence clinical interventions in AML should be suitably reformed to address and ablate both bulk, non-LSC leukemic blasts as well as LSC compartments, without disrupting normal hematopoiesis. We recently reported development of protein-vorinostat nanomedicine and demonstrated its remarkable anti-leukemic activity in AML patient samples (n=9) and AML cell lines (n=3).9 Notably NV selectively impaired clonogenic proliferation of leukemic bone marrow cells (BMC), while largely sparing healthy BMC indicating a strong possibility of destabilizing the LSC fraction. Recently,

decitabine

(Dacogen®,

Astex/Eisai),

DNMT

inhibitor

FDA-approved

for

myelodysplastic syndrome incited clinical attention after demonstrating significantly improved complete remission rates in a large randomized international phase III study, comparing decitabine to supportive care and cytarabine in elderly AML patients. However, difference in survival rates failed to reach significance, and FDA approval was not granted to decitabine for the treatment of AML.

10-11

Currently, intense research is focused on further defining subgroups of elderly AML patients who may derive greater benefit from decitabine therapy, in combination with other low-intensity active agents 2 ACS Paragon Plus Environment

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for AML.12-13 In view of these, we designed our specific study objectives to investigate (1) anti-LSC effect of NV on CD34+ CD38- CD123+ fraction (2) myelosuppression and hemotoxicity potential of NV, and (3) possible synergism of lower concentrations of NV with decitabine, for a prospective more tolerable, and effective therapeutic regimen for elderly AML patients. NV was synthesized using a modified coacervation technique, reported earlier by our group, 9 and characterized using scanning electron microscopy (SEM) and dynamic light scattering (DLS) techniques. Peripheral blood/bone marrow samples were obtained after written informed consent from AML patients and healthy donors upon approval from Institutional Ethics Committee, Amrita Institute of Medical Sciences and Research Centre, Kochi, Kerala, India. Mononuclear cells were isolated using standard density gradient/centrifugation method. Isolated cells were suspended in serum free StemSpan™ H3000 medium supplemented with StemSpan™ CC100 cytokine cocktail. For flow cytometric enumeration of leukemic stem/progenitor cell (LSPC) population, initially viable healthy cell fraction was selected based on forward scatter-side scatter (FSC-SSC) gating to form the P1 population. From P1, CD123+ cell fraction was gated as P2 and CD34+ CD38- cells as P3 populations, respectively. From P2 and P3 fractions, P4, constituting the target viable 7-AAD- CD34+ CD38- CD123+ LSPC fraction was derived. For quantifying viability of LSPC, cells were incubated with 10 µg/mL 7amino-actinomycin D (7-AAD). The anti-LSPC effect was analyzed by monitoring absolute numbers (considering number of events in respective gates = number of cells in respective gates) of viable LSPC in P4 quadrant (7-AAD- CD34+ CD38- CD123+) before (t=0) and after treatment (t=72 h) with 1 µM NV. Colony forming unit assay was performed to assess the clonogenic proliferation capability of NV treated LSPC and healthy BMC. Hemolytic potential of NV was analyzed spectrophotometrically by using Soret band based absorption of free hemoglobin in blood plasma at 415 nm.14-15 Lymphocyte proliferation and immunosuppression analysis were carried out to investigate for probable immunogenic or immunosuppressive properties of NV. Possible synergism of NV with decitabine was evaluated by treating patient sample derived cells with 0.1 µM NV and varying concentrations of decitabine (0.1-1 µM) simultaneously, for 72 hours. Cell viability was determined using MTT assay. 3 ACS Paragon Plus Environment


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Figure 1A shows the SEM image of spherical particles of NV measuring ~ 95 nm in size, and the DLS data (Figure 2A, inset) showing mean hydrodynamic diameter of 102 ± 8 nm (Figure 1A, inset) and a polydispersity index of 0.064. Nanoformulation exhibited a zeta potential of -31.08 mV, indicative of excellent stability. The clinical validity of NV induced toxicity was evaluated in CD34+ CD38- CD123+ LSC from two elderly AML patients and in normal BMC derived from healthy stem cell donor. Clinical characteristics of the patients at the time of sampling are summarized in Table 1.

Patient

Age/Gender

Sample

sample

Diagnosis with

Blasts*

FAB class

(%)

Cytogenetics*

Clinical status*

PS1

68/M

Bone marrow

M4 AML

81.2

None detected/46, XY

Refractory to decitabine

PS2

72/F

Bone Marrow

M2 AML

92

None detected/46, XX

Refractory to decitabine

*

at the time of sampling

Table 1. Clinical characteristics of patient samples, PS1 and PS2, at the time of sampling.

Figure 1B is a schematic diagram depicting the gating strategy employed for the enumeration of viable LSC. 16 Figure 1C and D shows the effect of NV on patient sample 1 (PS1) and 2 (PS2) respectively. Interestingly, NV was shown to completely eradicate the LSC population in PS1, since there were practically no viable cells in the P4 quadrant representing 7-AAD- CD34+ CD38- CD123+ fraction (Figure 1C, bottom panel). In PS2, after 72 hours of NV treatment, only 2 viable events were registered in the P4 quadrant (Figure 1D, bottom panel). LSC fraction was unaffected by treatment with bare albumin nanoparticles (vehicle control: Supporting Information Figure S1) for 72 hours. Since a near-absolute wipe-off of cells were observed in P1 populations of both PS1 (blast % = 81.2) and PS2 (blast % = 92) samples at t=72 h, it could be interpreted that NV exerted its effect on both


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blast and LSC fractions, effectively ablating both bulk and leukemic stem/progenitor cell compartments.

Figure 1. (A) SEM image showing spherical NV particles of ~ 95 nm. Inset: Hydrodynamic diameter (DLS) - 102 ± 8 nm. NV registered a zeta potential of -31.08 mV. (B) Schematic diagram of the gating strategy employed for the enumeration of viable LSC. Effect of NV on the viability of CD34+ CD38CD123+ LSC from (C) patient sample 1 (PS1), and (D) patient sample 2 (PS2). In sections C and D, upper



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panel: isotype controls; middle panel: number of cells before NV treatment (t=0h); bottom panel: number of cells after NV treatment (t=72h).

Although complete remissions have been reported in clinical trials, on account of lack of sustained anti-leukemic effect, vorinostat have not been approved for AML. The free drug faces shortcomings of hydrophobicity (0.2 mg/mL), low permeability (log partition coefficient of 1.9), and sub-optimal pharmacokinetics including low bioavailability (43%), extensive serum clearance and a short elimination half-life (~ 2 hours).17

We speculate that NV, intended for intravenous

administration, could effectively address the above limitations and with its remarkable cellular uptake and sustained drug release profiles could prolong the anti-leukemic effect compared to transient effect of the free drug.9 It could be inferred from the observed results that with well-preserved chemical stability and molecular activity, lower concentrations of encapsulated drug could efficiently exert its pleiotropic effect on the LSC effectively killing them.18 To validate the flow cytometry data, the effect of NV on the clonogenic growth potential of LSC was evaluated using colony forming unit (CFU) assay. This assay is also employed for screening anti-cancer agents for their neutropenia inducing potential. Chemotherapy induced neutropenia (CIN; characterized by extremely low level of neutrophils) and its complications are major dose-limiting toxicities of leukemic patients undergoing chemotherapy, and is associated with substantial morbidity, mortality, and economic burden.19 Figure 2A and C shows regular colony formation in untreated leukemic BMC from PS1 and PS2, respectively. Interestingly, 1 µM NV treatment completely disrupted colony formation in leukemic BMC as evident from virtual absence of colonies in NV treated PS1 and PS2 samples (Figure 2B and D). Conversely, clonogenic growth pattern of healthy BMC was not affected upto 5 µM NV (Figure 2F). Healthy BMCs treated with 5 µM NV were shown to produce CFUG and CFU-GM colonies. Figure 2G shows the graphical representation of median number of CFUs formed in untreated, and NV treated healthy and leukemic BMC from patients 1 and 2, after 14 days. 6 ACS Paragon Plus Environment

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Figure 2. Effect of NV on the clonogenic proliferation of (A & B) PS1 (C & D) PS2 (E & F) healthy bone marrow cells. (G) Graphical representation of number of CFUs formed in control, and NV treated healthy BMC and leukemic BMC from patients 1 and 2, after 14 days. ‘X’ denotes zero CFU in NV treated samples. (H) Hemolysis data of free and nano-vorinostat. Inset: Photograph of the whole blood treated with Triton, 1 µM, 5 µM and 10 µM NV. Scanning electron micrograph of RBC treated with (I) PBS and (J) 10 µM NV. Vorinostat has been documented to raise intracellular reactive oxygen species (ROS) levels, in addition to inhibiting HDAC

20

. Moreover, recent reports have also shown vorinostat to inactivate

thioredoxin, (ubiquitous ROS scavenger), permitting the drug induced ROS levels to elevate 21. Healthy cells are reported to accumulate thioredoxin upon treatment with HDAC inhibitors. Therefore, ROS generated by HDAC inhibitors in normal cells gets neutralized by thioredoxin, whereas cancer cells 7 ACS Paragon Plus Environment


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with inactivated thioredoxin, succumb to oxidative stress 22. Therefore, differences in the acetylome and expression levels of thioredoxin in normal cells could be attributed to the relative resistance of healthy HSC to vorinostat mediated cytotoxicity. Thus, NV at a low concentration of 1 µM exerting selective cytotoxicity against LSC, without presenting any myelosuppressive activity upto 5 µM, exhibits a highly desirable trait in contrast to currently administered myeloablative chemoregimens.

We subsequently analyzed the hemocompatibility of the nanoformulation compared to free vorinostat, by probing for any hemolytic, immunogenic or immuosuppressive potential of NV. Figure 2H revealed that both free drug and NV did not induce any hemolysis up to 10 µM. Plasma of 10 µM NV treated whole blood samples remained clear without any leakage of free hemoglobin (Figure 2H inset). Scanning electron micrographs (SEM) revealed that 10 µM NV treated erythrocytes retained intact morphology (Figure 2J) similar to those treated with PBS (Figure 2I). Similarly, lymphocytes treated with NV alone did not show any spontaneous proliferation (Figure 3A) up to 10 µM, indicating that the nanosystem did not induce any immunogenicity.


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Figure 3. (A) Immunogenicity studied by analyzing cell viability of lymphocytes upon exposure to different concentrations of free vorinostat and NV for 72 hours in comparison with PHA-M treatment (B) Immunosuppression studied by cell viability analysis of lymphocytes exposed to mixture of PHA-M + FV/NV for 72 hours. Optical microscopic images of lymphocytes treated with (C) PBS (D) 10 µM NV (E) PHA-M and (F) PHA-M + 10 µM NV.

To investigate for any immunosuppressive affect, proliferation of lymphocytes were analyzed in the presence of both phytohemagglutinin (PHA-M; mitogen) and NV. Lymphocytes treated with combination of PHA-M and varying concentrations of NV showed proliferation patterns similar to cells treated with PHA-M alone, suggesting that NV did not exhibit any immunosuppressive potential by subduing lymphocyte proliferation activated by the mitogen (Figure 3B). SEM of PBS (Figure 3C) and 10 µM NV (Figure 3D) treated lymphocytes revealed discrete cells without significant clumping. Whereas, lymphocytes treated with PHA-M and PHA-M + 10 µM NV showed PHA-M mediated agglutination (Figure 3E and F). Interestingly, both free drug and NV displayed almost similar hemocompatibility profiles. Briefly, NV without exhibiting any substantial myelosuppressive, hemolytic, immunogenic or immunosuppressive potential projects appreciable hemocompatibility. Considering the anti-leukemic activity of NV against both blasts and LSC, next study objective was to investigate its synergism with the hypomethylating agent, decitabine. Based on positive indications from a randomized international phase III study, decitabine was approved by the European Commission as a frontline therapy for untreated high-risk elderly AML patients.13 However, owing to lack of significant survival differences, FDA approval was not granted for the same, but continues to be used off-label.

10-11

In view of widely observed decitabine resistance, and ongoing research on

combining decitabine with other low-intensity active agents for AML, we surveyed for synergistic effects of simultaneous treatment of 0.1 µM NV with varying concentrations of decitabine (0.1 – 1 µM), in PS1 and PS2 for 72 hours. Both patients showed refractoriness to decitabine treatment. Figure 9 ACS Paragon Plus Environment


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4A and C showed dose dependent toxicity of free decitabine towards PS1 and PS2. However, no significant toxicity was observed at lower test concentrations. 0.1 µM NV alone too did not exert any considerable toxicity (green bars in Figure 4B and D). Remarkably, combination of 100 nM decitabine and 0.1 µM NV registered 76% toxicity in PS1 and 96% in PS2, which further showed absolute toxicity with higher concentrations of decitabine resulting in 100% cytotoxicity, in both patient samples (Figure 4B and D). Eventhough number of patient samples were limited (n=2), the observed trend point towards the excellent synergistic lethality of decitabine and NV towards refractory AML cells. Several studies attest to the fact that compared to genetic lesions in AML, epigenetic lesions appear to be more frequent and recurrent, and majority of the AML cases exhibit both aberrant histone deactylation and DNA methylation profiles 23. Considering these atypical DNA methylation (in multiple functionally relevant genes including, but not limited to p15, p73, E-cadherin, ID4, and RARβ2), 23-24 and histone deacetylation patterns (direct result of chromosomal translocations: AML1ETO, PML-RARα etc)25 in AML, therapeutic outcomes arising from a ‘dual epigenetics targeted hit’ using a combination of HDAC inhibitor, vorinostat, and a DNMT inhibitor, decitabine, would definitely look more promising. Clinical studies have underlined these epigenetic therapies to have acceptable safety and efficacy profiles. 26 Most importantly, absolute cell-kill was observed in both patient sample cells, indicative of the promise of this treatment regimen. This strategy shows promise in the setting of minimal residual disease (MRD) requiring prolonged treatment, and could provide improved therapeutic outcomes in elderly patients, those who are judged unfit for intensive chemotherapy with cytarabine and daunorubicin.


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Figure 4. Cell viability data showing the effect of decitabine alone and combination of decitabine with 0.1 µM NV in (A & B) PS1 (C & D) PS2. In the context of functional and clinical significance of the primitive leukemic cell population in therapeutic failure and relapse of AML, NV showing effective ablation of blast and LSC populations, without inducing any myelosuppresion or hemotoxicity can be considered as a promising candidate for further pre-clinical evaluation and translation. In addition, the observed synergism between NV and decitabine in refractory elderly patient samples provides an optimistic choice of a more tolerable and effective dual epigenetic targeted therapeutic approach for AML, over current cytotoxic, myeloablative chemotherapy.



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ASSOCIATED CONTENT Supporting Information The following file is available free of charge on the ACS Publications website. File name: Detailed experimental methods and supporting data (PDF).

AUTHOR INFORMATION Corresponding Authors Prof. Manzoor Koyakutty, PhD *E-mail: [email protected] Prof. Shantikumar Nair *E-mail: [email protected] Notes All authors have given approval to the final version of the manuscript and declare no competing financial interest, disclosure or conflicts of interest.

ACKNOWLEDGEMENTS The work was supported by Department of Biotechnology (DBT), Government of India, under the project ‘In silico design, development, nanotoxicology and preclinical evaluation of theragnostic cancer nanomedicine Phase-II’ (BT/PR14920/NNT/28/503/2010). Dr. Chandran thanks Council of Scientific and Industrial Research (CSIR), Government of India, for Senior Research Fellowship. Authors are grateful to Mrs. Sreerekha PR, for her technical assistance in the work.


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For Table of Contents Use Only

Protein nanomedicine exerts cytotoxicity towards CD34+ CD38- CD123+ leukemic stem cells

Parwathy Chandrana, c, PhD, Keechilat Pavithranb, MD, DM, Neeraj Sidharthanb, MD, DM, Abhilash Sasidharana,c, PhD, Shantikumar Naira*, PhD, Manzoor Koyakuttya*, PhD a

Amrita Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham University, Kochi, Kerala, India

b

Department of Medical Oncology, Amrita Institute of Medical Sciences and Research Centre, Kochi, Kerala, India c

Nanotechnology Innovation Center of Kansas State (NICKS), Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas, USA



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For Table of Contents Use Only 81x48mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Protein Nanomedicine Exerts Cytotoxicity toward CD34+

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