Modeling of Chronic Myeloid Leukemia: An Overview of In Vivo Murine and Human Xenograft Models

Table 1

Overview of in vivo CML models.

(a)

Mouse models	Method	Phenotype of leukemia	Features	References
Conventional transgenic	Synthetic BCR-v-ABL driven by Eμ or promoter/enhancer of MPSV LTR	Pre-B and T lymphomas (3/12 with EμVh vector), T lymphomas (1/3 with MPSV LTR vector)	BCR-v-ABL possesses oncogenic capability in vivo.; Synthetic BCR-v-ABL was not similar to original hybrid protein. Increased embryonic lethality	[27]
Conventional transgenic	p190 BCR-ABL gene driven by delta metallothionein-1 promoter	Myeloid leukemia (2/8 mice) and lymphoid leukemia (6/8 mice) between 10 and 58 days of birth	Follow-up studies with bigger cohort developed transplantable ALL/lymphoma	[28, 29]
Conventional transgenic	p210 BCR-ABL gene driven by delta metallothionein-1 promoter	T cell leukemia	The tumorigenicity of p210 BCR-ABL chimeric gene products is specific for the hematopoietic cells	[30]
Conventional transgenic	p210 BCR-ABL gene driven by the mouse tec promoter	ALL (2/5 founder mice developed), MPDs in transgenic progeny with 4–8 months of latency	Transgenic progeny of one founder mice exhibited MPD with fundamental features of CML. Secondary mice showed excessive proliferation of myeloid and megakaryocytic cells; however, they succumbed to progressing anemia	[31]
Conditional transgenic	Tet-off system: tTA driven by the MMTV-LTR promoter	Lethal B-ALL developed in 100% of mice within 3–11 weeks on withdrawal of tetracycline due to p210 BCR-ABL1 expression	MMTV-LTR promoter directed expression of tTA to B220^+ BM cells. Abolition of BCR-ABL1 expression led to apoptosis of leukemic cells and hence reversal of B-ALL phenotype. Reverted mice from one founder did succumb to ALL without BCR-ABL expression, possibly due to the acquisition of additional mutations	[32]
Conditional transgenic	Tet-off system: tTA driven by the SCL promoter	Neutrophilia, leukocytosis, and dissemination of myeloid cells into spleen, liver, and lymph nodes within 29–122 days upon tetracycline withdrawal due to p210 BCR-ABL1 expression.; 31% of mice progressed to B-ALL	SCLtTA/BCR-ABL expression model recapitulates many features of human CP-CML	[33]
Conditional transgenic	Overexpression of BMI1 by ubiquitin C promoter in a lentiral EGFP vector in BCR-ABL expressing CP-CML stem cells and progenitor using Scl/p210 BCR-ABL binary mouse model	Development of serially transplantable B-ALL with accumulation of BMI1/BCR-ABL^+ B-cells after 16 weeks of transplantation	BMI1 synergizes with BCR-ABL to transform chronic-phase SCL/p210 B-lymphoid progenitors but not HSCs or multipotent progenitors (MPPs) and imparts a proliferative advantage to induce serially transplantable B-ALL in vivo	[34]
Transgenic by homologous recombination	E14 ES cells to create in-frame fusion of p190 BCR-ABL with exon 1 of murine bcr in the presence or absence of the endogenous nonrearranged bcr allele	bcr-ABL/bcr^+ chimeric mice and 37/40 bcr-ABL/bcr^- developed B-ALL	bcr-ABL fusion gene does not require endogenous bcr allele to develop leukemia	[35]
Transduction/transplantation	Mu BM transduced with retroviral construct of p210 BCR-ABL expressed under control of MPSV myeloid cell-specific promoter	13/30 developed 3 distinct malignancies: CML, ALL, and macrophage-like tumor	Retrovirus mediated expression of p210 BCR-ABL demonstrates murine model system for CML	[5]
Transduction/transplantation	Mu BM of 5-fluorouracil; (5-FU) treated mice transduced with v-abl or p210 BCR-ABL retrovirus	>90% mice that received v-abl or BCR-ABL transduced cells died due to myelomonocytic leukemia, granulocytic leukemia, and pre-B-cell lymphoma	Both BCR-ABL and activated v-abl can induce similar malignancies. Integration site analyses allowed evaluation of clonality	[36]
Transduction/transplantation	Mu BM transduced with retroviral construct of p210 BCR-ABL under control of MSCV LTR promoter in the presence of SCF	All recipients came down with disease and displayed markedly elevated WBC counts with granulocyte predominance	Induction of murine CML in 100% of recipients with 4–6 weeks of latency.; All secondaries succumbed to lymphoid neoplasms.	[37]
Transduction/transplantation	5-FU treated Mu BM cells transduced with retroviral construct of p210 BCR-ABL expressed under control of MSCV promoter	Elevated WBC counts, majority of which were granulocytes but also included myeloblasts and basophils	Induction of transplantable myeloproliferative disease resembling CML.; Leukemic cells expressed excess IL3 and GM-CSF	[38, 39]
Transduction/transplantation	5-FU treated/untreated Mu BM cells transduced with retroviral construct of p210 BCR-ABL, p190 BCR-ABL, and p230 BCR-ABL expressed under control of MSCV promoter	CML-like syndrome when 5-FU treated donor cells were used.; Mixture of CML-like disease, B-ALL, and macrophage tumors when non-5-FU treated donor cells were used	All 3 forms of BCR-ABL induce identical CML-like syndrome in mice but p190 BCR-ABL had increased potency for induction of B-ALL	[40]
Transduction/transplantation	5-FU treated bone marrow cells with a retrovirus encoding; p210 BCR-ABL together with the XZ-miR-125b overexpressing; miR-125b or the control vector XZ were transplanted into lethally irradiated BALB/c recipient mice	50% of mice died of B-ALL, 42% with MPN, and 8% of mixed (myeloid and lymphoid) leukemia when transplanted with miR-125b plus BCR-ABL-infected cells with median survival of 21 days as compared to 35 days in BCR-ABL transduced control group	miR-125b accelerates the oncogenicity of BCR-ABL in transplanted mouse model	[41]
Transduction/transplantation	B-ALL LDBM cells from specific gene-deleted murine models or WT mice and UCB CD34^+ were transduced with bicistronic; vectors expressing EGFP and p190-BCR-ABL (MSCV-p190-BCRABL) or only EGFP (MIEG3) and then cultured with IL-7 and SCF	>90% of recipient mice developed B-ALL in approximately 37 days characterized by B220, CD19^+, and CD43 B-cell progenitor population. Infiltration in other organs was also evident	Vav3 plays a crucial role in p190-BCR-ABL-mediated leukemogenesis, proliferation, and survival especially for the B-cell progenitor	[42]

(b)

Human models	Method	Phenotype of leukemia	Features	References
Xenograft; nude mice	Nude mice injected with K562	K562 grew as solid vascularized tumors	Tumor cells were triphoid and retined human chromosome markers	[43]
Xenograft; nude mice	SIA-nu/nu mice were injected with leukemic cell lines and primary patient sample	K562 formed solid tumor at challenged site without metastatic spread with mean latency of 10 days	6/8 leukemic cell lines and 5/18 primary neoplastic tumors induced serially transplantable solid soft mass	[44]
Xenograft; SCID mice	BM samples of CP-CML and BC-CML as well as cell lines; K562 and EM-2 were transplanted into CB-17 scid/scid mice	All mice injected with K562 as well as EM-2 or primary CP-CML and BC-CML samples by IV or IP engraft to give myeloblasts in BM, blood, and tumors in peritoneum	After initial growth in kidney capsule, myeloblasts were detected at varying levels in PB and BM.; Human myeloid and lymphoid leukemia cell lines showed distinct growth patterns.; Differences were also observed in engraftment of CP versus BC-CML primary patient samples	[45]
Xenograft; SCID mice	BM or PB samples obtained from CP-CML and BC-CML patients were injected by IV into sublethally irradiated 400 cGy SCID mice. Exogenous cytokines PIXY321 or c-kit ligand was injected IP	CP-CML and BC-CML patient sample showed 1–>10% engraftment with 30–60 days of latency in presence or absence of exogenous human cytokines	Multilineage engraftment and CD34^+ cells were maintained for more than 60 days after transplantation.; First evidence that both normal and leukemic CP-CML cells can engraft in SCID mice	[46]
Xenograft; NOD/SCID	MNCs from apheresis material from CML patient were IV transplanted into sublethally irradiated 300 cGy NOD/SCID mice. Preselected CD34^+ and CD34^− cells were also used for BM engrafted studies	≥1–84% multilineage engraftment observed in BM in 76% mice and only 66% of mice showed 16% predominantly T cell splenic engraftment. CML-like disease in BM and spleen. 39%  ±  5% leukemic engraftment in 25 mice having ≥9% BM engraftment was higher as compared to BCR-ABL engraftment in spleen	Higher engraftment in NOD/SCID mice using low cell dose compared to SCID mice	[47]
Xenograft; NOD/SCID	MNCs or CD34^+ enriched cells from BM or PB of 11 CP-CML patient were IV transplanted into sublethally irradiated 400 cGy NOD/SCID mice	25% of NOD/SCID recipients had 40–80% human cells, whereas only 3% SCID mice contained similar levels. Further, engrafted human cells had high proportion of leukemic cells along with CD34^+ cells	NOD/SCID mice allow greater engraftment and amplification of both normal and leukemic cells as compared to SCID mouse model	[48]
Xenograft; NOD/SCID	BV173 and PB MN cells from CP, AP, and BC CML patient samples were injected	Kinetics and extent of engraftment BP > AP > CP, although according to growth rate BP > AP ≥ CP	Kinetics of BM repopulation are different for CP, AP, and BC phase of CML	[49]
Xenograft; NOD/SCID	9 CP-CML patient samples with predominant LTC-IC were transplanted into sublethally irradiated 350 cGy NOD/SCID and NOD/SCID β2m^−/− mice	Consistent and durable engraftment was observed with reduced output of B cells and enhanced myelopoiesis with excessive production of erythroid, megakaryocytes, and BCR-ABL CD34^+ expressing IL-3 and G-CSF transcripts	No progressive disease phenotype was observed marking CP-CML phase of the disease	[8]
Xenograft; NOD/SCID	CB CD34^+ cells were transduced with MSCV based retroviral constructs for BCR-ABL and transplanted 0.2 to 0.3 million cells into each sublethally irradiated 350 cGy NOD/SCID and NOD/SCID β2m^−/− mice	BCR-ABL transduced cells produced increased ratio of myeloid to B-lymphoid cells with increase in erythroid and megakaryocytic cells. 4/28 mice developed an increased WBC count and/or splenomegaly after 5-6 months of latency	First ever model to describe the de novo generation of preleukemic cells by forced expression of BCR-ABL in human CD34^+ CB cells. Primary CD34^+ CB cells showed rapid and persistent deregulation and erythroid and megakaryocytic biased differentiation in vivo with occasional progression to an early stage of CML	[50]
Xenograft; NOD/SCID	CB CD34^+ cells were transduced with MSCV based retroviral constructs for BMI1 and BCR-ABL and transplanted only 0.46 to 0.38 million cells into each sublethally irradiated 3 Gy NOD/SCID mice	4/8 mice succumbed to [CD34^+ CD19^+] B-ALL in 16–22 weeks on transplantation of CD34^+ cells cotransduced with BMI/BCR-ABL and all secondaries came down with similar phenotypes within 8–12 weeks	Coexpression of BMI1 and BCR-ABL in CB CD34^+ cells is sufficient to induce transplantable B-ALL in NOD/SCID mice	[51]
iPSC	KBM7 cells were reprogrammed by retroviral transduction of OCT4, SOX2, c-MYC, and KLF-4	Teratoma formation and imatinib resistance were observed	The process of reprogramming KBM7 cell lines readily abolished BCR-ABL dependency which was restored by differentiation into hematopoietic lineage	[52]
iPSC	MNCs from BM of CP-CML patient sample were cultured with human SCF, IL-3, IL-6, and Flt3L for 2 days and transfected with episomal vectors by nucleofection	CP-CML iPSC lines generated exhibited features of pluripotent stem cells, exhibited complex karyotype, and differentiated into hematopoietic lineages	Transgene free CML iPSC lines can be obtained	[53]
iPSC	Regeneration of CML iPSCs from CD34^+ BM MNCs of CP-CML patient sample by retroviral vectors	DNA methylation pattern and gene expression profile of CML-iPSCs were different from those of original CML sample but were similar to normal iPSCs and human ES cells	Recapitulation of CP CML was shown in terms of that fraction of phenotypically immature cells which showed imatinib resistance although more differentiated cells recovered the sensitivity to imatinib	[54]