ALBERT

Description: Anaplastic large-cell lymphomas (ALCLs) bearing the t(2;5) translocation (ALK+ALCLs) are frequently characterized by skin colonization and associated with a poor prognosis. Using conditional transgenic models of anaplastic lymphoma kinase–positive (ALK+) lymphomas and human ALK+ALCL cell lines, in the present study, we show that high-mobility-group box-1 (HMGB-1), a proinflammatory cytokine, is released by ALK+ cells, and demonstrate extracellular HMGB-1–stimulated secretion of the IL-8 chemokine by HaCaT keratinocytes through the involvement of MMP-9, PAR-2, and the NF-κB pathway. Furthermore, we demonstrate that, in vitro, IL-8 is able to induce the invasiveness of ALK+ cells, which express the IL-8 receptors CXCR1 and CXCR2. In vitro and in vivo, HMGB-1 inhibition achieved by glycyrrhizin treatment led to a drastic reduction in ALK+ cell invasiveness. The pathophysiological relevance of our observations was confirmed by demonstrating that the HMGB-1 and IL-8 receptors are expressed in ALK+ALCL biopsies. We have also shown that IL-8 secretion is correlated with leukemic dissemination of ALK+ cells in a significant number of patients. The results of the present study demonstrate for the first time a relationship among the pro-inflammatory mediators HMGB-1, MMP-9, PAR-2, and IL-8. We propose that these mediators create a premetastatic niche within the skin, thereby participating in ALK+ lymphoma epidermotropism.

Description: The targeted inactivation of oncogenes may be a specific and effective treatment for cancer. However, because human cancers are the consequence of multiple genetic changes, the inactivation of one oncogene may not be sufficient to cause sustained tumor regression. Moreover, cancers are genomically unstable and may readily compensate for the inactivation of a single oncogene. Here we confirm by spectral karyotypic analysis that MYC-induced hematopoietic tumors are highly genetically complex and genomically unstable. Nevertheless, the inactivation of MYC alone was found to be sufficient to induce sustained tumor regression. After prolongedMYC inactivation, some tumors exhibited a distinct propensity to relapse. When tumors relapsed, they no longer required the overexpression of MYC but instead acquired novel chromosomal translocations. We conclude that even highly genetically complex cancers are reversible on the inactivation of MYC, unless they acquire novel genetic alterations that can sustain a neoplastic phenotype.

Description: NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) and TPM3-ALK (nonmuscular tropomyosin 3-anaplastic lymphoma kinase) are oncogenic tyrosine kinases implicated in the pathogenesis of human ALK-positive lymphoma. We report here the development of novel conditional mouse models for ALK-induced lymphomagenesis, with the use of the tetracycline regulatory system under the control of the EμSRα enhancer/promoter. The expression of either oncogene resulted in the arrest of the differentiation of early B cells and lymphomagenesis. We also observed the development of skin keratoacanthoma lesions, probably because of aberrant ALK expression in keratinocytes. The inactivation of the ALK oncogene on doxycycline treatment was sufficient to induce sustained regression of both hematopoietic tumors and skin disease. Importantly, treatment with the specific ALK inhibitor (PF-2341066) also reversed the pathologic states, showing the value of these mouse models for the validation of ALK tyrosine kinase inhibitors. Thus, our results show (1) that NPM-ALK and TPM3-ALK oncogenes are sufficient for lymphoma/leukemia development and required for tumor maintenance, hence validating ALK as potentially effective therapeutic target; and (2) for the first time, in vivo, the equal tumorigenic potential of the NPM-ALK and TPM3-ALK oncogenic tyrosine kinases. Our models offer a new tool to investigate in vivo the molecular mechanisms associated with ALK-induced lymphoproliferative disorders.

Description: With the use of microarray gene-expression profiling, we analyzed a homogeneous series of 32 patients with systemic anaplastic large-cell lymphoma (ALCL) and 5 ALCL cell lines. Unsupervised analysis classified ALCL in 2 clusters, corresponding essentially to morphologic subgroups (ie, common type vs small cell and “mixed” variants) and clinical variables. Patients with a morphologic variant of ALCL had advanced-stage disease. This group included a significant number of patients who experienced early relapse. Supervised analysis showed that ALK+ALCL and ALK− ALCL have different gene-expression profiles, further confirming that they are different entities. Among the most significantly differentially expressed genes between ALK+ and ALK− samples, we found BCL6, PTPN12, CEBPB, and SERPINA1 genes to be overexpressed in ALK+ ALCL. This result was confirmed at the protein level for BCL-6, C/EBPβ and serpinA1 through tissue microarrays. The molecular signature of ALK− ALCL included overexpression of CCR7, CNTFR, IL22, and IL21 genes but did not provide any obvious clues to the molecular mechanism underlying this tumor subtype. Once confirmed on a larger number of patients, the results of the present study could be used for clinical and therapeutic management of patients at the time of diagnosis.

Description: Cancer is caused by genetic events that result in the activation of oncogenes or the inactivation of tumor suppressor genes. Using the Tetracycline system, our laboratory has generated a transgenic mouse model in which MYC is conditionally overexpressed in hematopoietic cells, allowing us to turn MYC expression on and off at will. We have previously demonstrated that the inactivation of this single oncogene in an established and even highly invasive and metastatic lymphoma is sufficient to reverse cancer, suggesting that MYC may be an effective therapeutic target. In a variety of conditional oncogene models, we and others have found that tumor regression following oncogene inactivation involves similar phenomena, including cell cycle arrest, apoptosis, and differentiation of the tumor cells. The similarity in the regression process across a variety of different oncogenes and types of cancer strongly suggests the existence of a common signaling pathway following oncogene inactivation, which culminates in the cessation of cell proliferation and the induction of apoptosis and differentiation. Recently, we have demonstrated that MYC activation disrupts the repair of DNA breaks and results in genomic instability. We speculated that MYC inactivation may cause tumor regression by restoring the ability of tumor cells to recognize that they are genomically damaged, which could subsequently lead to the cell cycle arrest, differentiation and apoptosis that is generally observed. Indeed, we now report that upon MYC inactivation, tumor cells activate DNA damage signaling pathways and begin to repair their DNA breaks. We have found evidence for activation of a functional DNA damage response both by immunofluorescent staining for phosphorylated ATM and Mre11, which demonstrates foci formation following MYC inactivation; and by the Comet assay, which shows a quantitative decrease in severity of DNA breaks following MYC inactivation. Our results suggest that MYC inactivation may induce tumor regression at least in part through the restoration of a DNA damage checkpoint response.

Description: MYC overexpression is thought to induce tumorigenesis through a variety of different mechanisms including the induction of proliferation, inhibition of differentiation and disruption of genomic stability. Conventional transgenic systems that have been used to study the role of oncogenes in tumorigenesis continuously overexpress transgenes and hence preclude the investigation of the initial and age specific consequences of oncogene activation. To investigate the developmental specific consequences of MYC overexpression in the pathogenesis of lymphoma in vivo, we used transgenic mice in which the MYC proto-oncogene is conditionally regulated via the Tetracycline Regulatory System (Tet system). The ability of MYC to induce lymphomagenesis was found to be inversely correlated with the age of the host at the time of MYC activation. When MYC was activated constitutively, the mean time until tumor development was 13 weeks. When MYC was activated at increasing developmental ages of 3 and 5 weeks, identical tumors arose, but with an increased mean tumor latency. When MYC was activated at 7 weeks, an age equivalent to an adult mouse, no tumors developed even after 40 weeks of observation. However, we found that in adult mice, if both MYC and BCL2 are overexpressed at 7 weeks of age, mice succumbed to lymphoma with a mean latency of 27 weeks. Surprisingly, we could not find evidence that MYC overexpression induces apoptosis or that BCL2 overexpression reduced apoptosis. We conclude that the ability of MYC to induce lymphomagenesis is highly dependent on the developmental context. MYC and BCL2 cooperate to permit tumorigenesis in adult mice. Since lymphoma occurs generally as a disease in adults, this suggests that previous reports may have greatly overestimated the ability of MYC activation to induce tumorigenesis and underestimated the potential cooperation between MYC and BCL2 oncogenes. Our results also suggest that BCL2 may cooperate with MYC to induce lymphomagenesis through additional mechanisms other than preventing MYC from inducing apoptosis.

Description: The blood is a perpetually renewing tissue seeded by a rare population of adult bone marrow hematopoietic stem cells (HSC). During steady-state hematopoiesis, the HSC population is relatively quiescent but constantly maintains a low numbers of cycling cells that differentiate to produce the various lineage of mature blood cells. However, in response to hematological stress, the entire HSC population can be recruited into cycle to self-renew and regenerate the blood-forming system. HSC proliferation is therefore highly adaptative and requires appropriate regulation of cell cycle progression to drive both differentiation-associated and self-renewal-associated proliferation, without depletion of the stem cell pool. Although the molecular events controlling HSC proliferation are still poorly understood, they are likely determined, at least in part, by regulated expression and/or function of components and regulators of the cell cycle machinery. Here, we demonstrate that the long-term self-renewing HSC (defined as Lin−/c-Kit+/Sca-1+/Thy1.1int/Flk2−) exists in two distinct states that are both equally important for their in vivo functions as stem cells: a numerically dominant quiescent state, which is critical for HSC function in hematopoietic reconstitution; and a proliferative state, which represents almost a fourth of this population and is essential for HSC functions in differentiation and self-renewal. We show that when HSC exit quiescence and enter G1 as a prelude to cell division, at least two critical events occur: first, during the G1 and subsequent S-G2/M phases, they temporarily lose efficient in vivo engraftment activity, while retaining in vitro differentiation potential; and second, they select the particular cell cycle proteins that are associated with specific developmental outcomes (self-renewal vs. differentiation) and developmental fates (myeloid vs. lymphoid). Together, these findings provide a direct link between HSC proliferation, cell cycle regulation and cell fate decisions that have critical implications for both the therapeutic use of HSC and the understanding of leukemic transformation.

Description: The hallmark (8:14) translocation of Burkitt’s lymphoma causes MYC overexpression, a primary event in the development of Burkitt’s lymphoma. While Burkitt’s lymphoma comprises nearly one third of childhood malignancies, it is rare in adults. Among many explanations is the possibility that the capacity for a lymphocyte to undergo malignant transformation by MYC activation is influenced by factors related to the developmental state of the host. To address this possibility, we used transgenic mice in which the MYC proto-oncogene is conditionally regulated via the Tetracycline Regulatory System (Tet system). When MYC was activated in cohorts of mice less than 3 weeks of age, mice universally succumbed to lymphoma within 23 weeks. However, when MYC was activated in adult mice (over 7 weeks of age) the mice failed to develop lymphoma within 50 weeks. We were not able to detect differences in the effects of MYC on the proliferation or apoptosis in lymphocytes from adult versus neonatal hosts. We next explored whether the concomitant activation of MYC with a second oncogene could induce lymphomagenesis in adult mice. When we conditionally induced both MYC and BCL2 or MYC and RAS in adult hosts, mice succumbed to lymphoma with a mean latency of 24–27 weeks. Our results suggest that in adult hosts, concurrent activation of at least two oncogenes is required for MYC to initiate tumorigenesis.

Description: Haematopoiesis is a tightly orchestrated process where a pool of hematopoietic stem and progenitor cells (HSPCs) with high self-renewal potential can give rise to both lymphoid and myeloid lineages. The HSPCs pool is reduced with ageing resulting in few HSPC clones maintaining haematopoiesis thereby reducing blood cell diversity, a phenomenon called clonal haematopoiesis. Clonal expansion of HSPCs carrying specific genetic mutations leads to increased risk for haematological malignancies. Therefore, it comes as no surprise that hematopoietic tumours develop in higher frequency in elderly people. Unfortunately, elderly patients with leukaemia or lymphoma still have an unsatisfactory prognosis compared to younger ones highlighting the need to develop more efficient therapies for this group of patients. Growing evidence indicates that macroautophagy (hereafter referred to as autophagy) is essential for health and longevity. This review is focusing on the role of autophagy in normal haematopoiesis as well as in leukaemia and lymphoma development. Attenuated autophagy may support early hematopoietic neoplasia whereas activation of autophagy in later stages of tumour development and in response to a variety of therapies rather triggers a pro-tumoral response. Novel insights into the role of autophagy in haematopoiesis will be discussed in light of designing new autophagy modulating therapies in hematopoietic cancers.