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In anaplastic large cell lymphoma, the ALK gene at 2p23 is known to be fused to NPM, TPM3, TPM4, TFG, ATIC, CLTC, MSN, and ALO17. All of these translocations result in the expression of chimeric ALK transcripts that are translated into fusion proteins with tyrosine kinase activity and oncogenic properties. We report a case showing a restricted cytoplasmic staining pattern of ALK and a novel chromosomal abnormality, t(2;22)(p23;q11.2), demonstrated by fluorescence in situ hybridization analysis. The result of 5′ RACE analysis showed that the ALK gene was fused in-frame to a portion of the non-muscle myosin heavy chain gene, MYH9. Nucleotide sequence of the MYH9-ALK chimeric cDNA revealed that the ALK breakpoint was different from all those previously reported. It is localized in the same exonic sequence as MSN-ALK, but 6 bp downstream, resulting in an in-frame fusion of the two partner proteins. In contrast to the previously reported ALK fusion proteins, MYH9-ALK may lack a functional oligomerization domain. However, biochemical analysis showed that the new fusion protein is tyrosine phosphorylated in vivo but seems to lack tyrosine kinase activity in vitro. If further investigations confirm this latter result, the in vivo tyrosine phosphorylation of MYH9-ALK protein could involve mechanisms different from those described in the other ALK hybrid proteins. © 2003 Wiley-Liss, Inc.

Leukemia-initiating/repopulating cells (LICs), also named leukemic stem cells, are responsible for propagating human acute leukemia. Although they have been characterized in various leukemias, their role in T-cell acute lymphoblastic leukemia (T-ALL) is unclear. To identify and characterize LICs in T-ALL (T-LIC), we fractionated peripheral blood cell populations from patient samples by flow cytometry into three cell fractions by using two markers: CD34 (a marker of immature cells and LICs) and CD7 (a marker of early T-cell differentiation). We tested these populations in both in vitro culture assays and in vivo for growth and leukemia development in immune-deficient mice. We found LIC activity in CD7(+) cells only as CD34(+)CD7(-) cells contained normal human progenitors and hematopoietic stem cells that differentiated into T, B lymphoid and myeloid cells. In contrast, CD34(+)CD7(+) cells were enriched in LICs, when compared with CD34(-)CD7(+) cells. These CD34(+)CD7(+) cells also proliferated more upon NOTCH activation than CD34(-)CD7(+) cells and were sensitive to dexamethasone and NOTCH inhibitors. These data show that CD34 and CD7 expression in human T-ALL samples help in discriminating heterogeneous cell populations endowed with different LIC activity, proliferation capacity and responses to drugs.

Optimized gene transfer into human primary leukemic T cell with NOD-SCID/leukemia-initiating cell activity

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a chimeric protein expressed in a subset of cases of anaplastic large cell lymphoma (ALCL) for which constitutive expression represents a key oncogenic event. The ALK signaling pathway is complex and probably involves functional redundancy between various signaling substrates of ALK. Despite numerous studies on signaling mediators, the molecular mechanisms contributing to the distinct oncogenic features of NPM-ALK remain incompletely understood. The search for additional interacting partners of NPM-ALK led to the discovery of AUF1/hnRNPD, a protein implicated in AU-rich element (ARE)-directed mRNA decay. AUF1 was immunoprecipitated with ALK both in ALCL-derived cells and in NIH3T3 cells stably expressing NPM-ALK or other X-ALK fusion proteins. AUF1 and NPM-ALK were found concentrated in the same cytoplasmic foci, whose formation required NPM-ALK tyrosine kinase activity. AUF1 was phosphorylated by ALK in vitro and was hyperphosphorylated in NPM-ALK-expressing cells. Its hyperphosphorylation was correlated with increased stability of several AUF1 target mRNAs encoding key regulators of cell proliferation and with increased cell survival after transcriptional arrest. Thus, AUF1 could function in a novel pathway mediating the oncogenic effects of NPM-ALK. Our data establish an important link between oncogenic kinases and mRNA turnover, which could constitute a critical aspect of tumorigenesis.

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells, we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells, whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover, long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage, strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether, these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors, and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis.

Recent efforts have been made to isolate molecular targets that could explain different outcome between histological subtypes of lymphomas and to understand the molecular mechanisms underlying oncogenic events. Using the SSH technique, we compared the transcriptome of 2 cases of Al.K + and ALK - anaplastic large cell lymphoma (ALCL) and of 2 cases of classical Hodgkin's lymphoma (cHL) with opposite behavior. Regarding ALCL, we showed that ALK-positive tumors overexpressed genes involved in different signaling pathways such as activation or signaling of T-cells, regulation of apoptosis, phospholipase Cγ and phosphatidyl inositol-3 Kinase. In addition, the characterization of a specific molecular signature may be of clinical relevance since ALK + tumors generally have a better prognosis than ALK - ones. Similar problems of differential prognosis is observed in cases of cHL, which in addition, may be morphologically and immunologically indistinguishable. Therefore, we applied the same SSH technique to 2 cHL samples from patients with favorable and poor outcome, respectively. Forty-four cDNAs were significantly overexpressed in the poor outcome case. In addition to the defender against death cell I (DAD1) gene, overexpressed clones corresponded mostly to expressed sequence tags (ESTs). Interestingly, the present study identifies new genes which may be involved in the pathogenesis and/or clinical outcome of cHL and deserve further investigations.

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Abstract 8 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors in which activating mutations of NOTCH1 occur in over 50% of cases and loss-of-function mutations in FBW7, which degrades activated NOTCH1, occur in 8-16% of cases. We and others have characterized c-MYC as a critical downstream transcriptional target of Notch signaling in this context, and loss of FBW7 also contributes here as intact FBW7 marks c-MYC protein for proteosomal degradation. Inhibition of Notch signaling in T-ALL cells by various means including small molecule gamma-secretase inhibitors (GSI) causes cell cycle/growth arrest and in some cases apoptosis, providing rationale for NOTCH1 as a therapeutic target. The tumor suppressor PTEN is also mutated or lost in up to 20% of T-ALL cases. PTEN activity converts PIP3 to PIP2, and thus loss of PTEN potentiates PI3K signaling. It was observed among human T-ALL cell lines that PTEN loss correlated with resistance to Notch inhibition with GSI, leading to the idea that constitutive PI3K/AKT signaling relieves T-ALL cells of their “addiction” to oncogenic Notch signaling. Since GSI and other NOTCH1 signaling inhibitors are presently under investigation as targeted therapeutic agents for T-ALL and other cancers including breast, brain, colorectal, pancreatic, and melanoma, this idea that PTEN loss could confer resistance to NOTCH1 inhibition raises concern that patients with PTEN-negative (or presumably PIK3CA-mutated) diseases may fail Notch inhibitor therapy. As the studies linking GSI-resistance/Notch-independence to PTEN loss were limited to established cell lines, we sought to address this issue using a well established and genetically defined mouse retroviral transduction/bone marrow transplantation model. Briefly, we generated primary murine T-cell acute leukemias using mutated NOTCH1 retroviruses on both wild-type and PTEN-null backgrounds and tested these tumors for response to GSI. Unexpectedly, we observed these primary murine leukemias to undergo growth arrest and cell size reduction with GSI treatment on both wild-type and PTEN-null backgrounds. We even observed wild-type background tumors which had lost PTEN spontaneously after either serial transplantation or extended culture in vitro to remain sensitive to GSI. Given that most cases of human T-ALL have also lost expression of p16INK4A/p14ARF tumor suppressors either by deletion or silencing, and since p16INK4A/p14ARF are critical regulators at the G1/S cell cycle checkpoint, we also generated primary murine tumors on a Pten, Ink4a/Arf double-null background. Even when both PTEN and Ink4a/Arf were deleted, we still observed the murine tumors to remain sensitive to GSI. To confirm these findings were not unique to murine tumors, we examined a panel of 13 primary human T-ALL cases including 7 PTEN-positive cases (6 of which were NOTCH1 mutated) and 6 PTEN-negative cases (4 of which were NOTCH1 mutated). We found 11 of these 13 cases to be GSI sensitive (6 PTEN-positive, 5 PTEN-negative) as illustrated by decreased proliferation and reduction in cell size. We found only 2 cases to be GSI resistant, including 1 PTEN-positive and 1 PTEN-negative. Our panel contained only one case with FBW7 mutation; this case was PTEN-positive and GSI-sensitive. Thus, in contrast to previous studies with established cell lines, we find in this study using primary human T-ALL samples and genetically defined primary murine leukemias that PTEN loss indeed does not confer resistance to Notch inhibition. Of note, other groups have postulated a role for FBW7 mutation in conferring resistance to Notch inhibition; however, we encountered only 2 GSI-resistant cases in our panel of primary human samples, neither of which was FBW7 mutated. In generating the murine Notch leukemias, we also had the opportunity to compare disease penetrance/latency and clonality among the various genetic backgrounds. We noted that PTEN loss was associated with accelerated disease onset and multiclonal tumors as compared to wild-type, whereas INK4A/ARF loss alone had no such effect. These findings suggest NOTCH1 activation and PTEN loss may collaborate in leukemia induction; however, technical caveats related to potentially enhanced retroviral transduction efficiency of PTEN-null bone marrow progenitors limit this conclusion, and thus more definitive experiments to address this issue are needed. Disclosures: No relevant conflicts of interest to declare.