Case 274 T lymphoblastic leukemia with t(5;14)(q35;q32) and lineage switch to acute myeloid leukemia Rebecca L. King, Gerald Wertheim, Michele E. Paessler The Children’s Hospital of Philadelphia Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA

Clinical History 13 year old boy 300 214 4.2 82% Blasts

Bone Marrow : April 2009

Flow Cytometry: April 2009 CD7+/CD4+ CD5+/CD2- CD14-/CD33- cCD3+/TdT+ MPO-

46,XY,t(5;14)(q35;q32),del(9)(p21.3),del(13)(q14.11q21.1)[7]. ish t(5;14)(IGH+;IGH-), del(9)(p16-) /46,idem,del(7)(p1?3p?21)[6]. /45,idem,dic(7;8)(p1?3;p11.2),del(10)(q22)[6]. ish dic(7;8)(7ptel-,7cen+,D7S486+,8cen+,MYC+) del(9)(p21)

Diagnosis T lymphoblastic leukemia t(5;14)(q35;q32) Del 9p

Clinical History 9.1 5.9 43 15% blasts Day 30 of DI: Blasts seen on peripheral smear. 9.1 15% blasts 5.9 43

Bone Marrow: 6 months after Dx MPO

Flow Cytometry: October 2009 CD14-/CD33+ CD15+ cCD3/TdT- MPO+

April 2009 October 2009 MPO MPO CD3 CD3

Therapy-related AML or Lineage switch from T-ALL to AML Diagnosis? Therapy-related AML or Lineage switch from T-ALL to AML

1. Cryptic t(5;14) detected by FISH with IGH probe 2. Del 9p der(15)t(11;15) del(7p) der(4) del(9)(p21) 67,XY,+Y,der(4)del(4)(q13.1)inv dup(4)(p12pter),del(7)(p11.2p14.1),+8,-9,del(9)(p21.3),-10,-11,-14,-15,der(15) t(11;15)(q13.1;q26),+19,+22[9].ish der(15)t(11;15)(MLL+,PML+) nuc ish 11q23(MLLx3)[89/100] nuc ish 15q22(PMLx2),17q21.1(RARAx3)[94/100] nuc ish 9p21(p16x1),9cen(CEP9x2)[90/100]

FISH: TLX3-BCL11b t(5;14)(q35;q32) present in 84% of nuclei Image courtesy of Rhett Ketterling, MD

Final Diagnosis T lymphoblastic leukemia with t(5;14)(q35;q32) and lineage switch to acute myeloid leukemia.

TLX3-BCL11b t(5;14)(q35;q32) 25% of pediatric T-ALL 5-10% adult T-ALL Single reported case of myeloid leukemia Overexpression of the orphan homeobox (HOX) proteins TLX1 and TLX3 represents the most frequent oncogenic event due to chromosomal translocation in human T-ALL HOX proteins in general, and TLX in particular, exert a repressive activity on transcriptional events during embryonic development As mentioned, an alternative and non-mutually- exclusive candidate for mediation of the maturation arrest might be BCL11b, recently reported to be a direct target downregulated by TLX1 (De Keersmaecker et al., 2010). Loss of BCL11b, a transcriptional repressor required for T lymphoid specification, leads, however, to a much earlier DN1/2 block (Ikawa et al., 2010; Li et al., 2000, 2004). knockdown of TLX1/TLX3 led to apoptosis, concomitant TCRa transcription and rearrangement, cell-maturation, and sTCRab+ expression prospective studies. Taken together, our results demonstrate that the maturation block observed in TLX+ T-ALLs is in large part due to ETS1-mediated TLX recruitment to the Ea core, leading to repression of Ea and blocked Va-Ja rearrangement. Failure to express a TCRa gene arrests development of ab-committed thymocytes around b-selection, when a variety of cell-proliferation signals are likely to be maintained, hence contributing to oncogenesis. This blockage can be overcome by TLX1/3 abrogation or by downstream TCRab expression within an appropriate cellular context. These observations have fundamental consequences both for targeted therapy in TLX+ T-ALLs and for the role of aberrant TCR expression in T lymphoid oncogene

Coincidence or biological relationship? Overexpression of the orphan homeobox (HOX) proteins TLX1 and TLX3 represents the most frequent oncogenic event due to chromosomal translocation in human T-ALL HOX proteins in general, and TLX in particular, exert a repressive activity on transcriptional events during embryonic development As mentioned, an alternative and non-mutually- exclusive candidate for mediation of the maturation arrest might be BCL11b, recently reported to be a direct target downregulated by TLX1 (De Keersmaecker et al., 2010). Loss of BCL11b, a transcriptional repressor required for T lymphoid specification, leads, however, to a much earlier DN1/2 block (Ikawa et al., 2010; Li et al., 2000, 2004). knockdown of TLX1/TLX3 led to apoptosis, concomitant TCRa transcription and rearrangement, cell-maturation, and sTCRab+ expression prospective studies. Taken together, our results demonstrate that the maturation block observed in TLX+ T-ALLs is in large part due to ETS1-mediated TLX recruitment to the Ea core, leading to repression of Ea and blocked Va-Ja rearrangement. Failure to express a TCRa gene arrests development of ab-committed thymocytes around b-selection, when a variety of cell-proliferation signals are likely to be maintained, hence contributing to oncogenesis. This blockage can be overcome by TLX1/3 abrogation or by downstream TCRab expression within an appropriate cellular context. These observations have fundamental consequences both for targeted therapy in TLX+ T-ALLs and for the role of aberrant TCR expression in T lymphoid oncogene

TLX3 Homeobox protein related to TLX1 Repressive activity on transcription during embryonic development Dadi et al. Cancer Cell, 2012. TLX1 and TLX3 suppress TCRα rearrangement via ETS1 accounting for the block in T-cell maturation seen in these T-ALLs. Knockdown of TLX1/3 leads to apoptosis, TCRα rearrangement and cell maturation Overexpression of the orphan homeobox (HOX) proteins TLX1 and TLX3 represents the most frequent oncogenic event due to chromosomal translocation in human T-ALL HOX proteins in general, and TLX in particular, exert a repressive activity on transcriptional events during embryonic development As mentioned, an alternative and non-mutually- exclusive candidate for mediation of the maturation arrest might be BCL11b, recently reported to be a direct target downregulated by TLX1 (De Keersmaecker et al., 2010). Loss of BCL11b, a transcriptional repressor required for T lymphoid specification, leads, however, to a much earlier DN1/2 block (Ikawa et al., 2010; Li et al., 2000, 2004). knockdown of TLX1/TLX3 led to apoptosis, concomitant TCRa transcription and rearrangement, cell-maturation, and sTCRab+ expression prospective studies. Taken together, our results demonstrate that the maturation block observed in TLX+ T-ALLs is in large part due to ETS1-mediated TLX recruitment to the Ea core, leading to repression of Ea and blocked Va-Ja rearrangement. Failure to express a TCRa gene arrests development of ab-committed thymocytes around b-selection, when a variety of cell-proliferation signals are likely to be maintained, hence contributing to oncogenesis. This blockage can be overcome by TLX1/3 abrogation or by downstream TCRab expression within an appropriate cellular context. These observations have fundamental consequences both for targeted therapy in TLX+ T-ALLs and for the role of aberrant TCR expression in T lymphoid oncogene

BCL11b Ikawa et al. Science, 2010. T cell Su et al. Blood, 2006. Significance of this gene thought to lie in downstream T-cell-specific cis-activating elements juxtaposed to TLX3 (leads to TLX3 upregulation) Ikawa et al. Science, 2010. BCL11b is a key checkpoint protein in T-cell development T/NK/ Myeloid T cell Multipotent HSC T/B/ NK/ Eryth/Meg B-cell BCL11b BCL11b is a critical gene in T cell development. Studies identified T cell specific cis activating elements downstream of BCL11b that are juxtaposed to the TLX3 locus as a result of the translocation. However, an intriguing finding about BCL11b was described in this 2010 study from Japan. IN this study they identified BCL11b as the critical protein at this checkpoint in T cell development in mice. It is likely that this is environmentally mediated, as decreased IL7 concentrations lead to upregulation of BCL11b which leads to downregulation of myeloid antigens. In addition, they describe the appearance of self renewing T cell progenitors in BCL11b-/- thymocytes in vitro suggests a mechanism for loss of function mutations as well as disruption of this gene to lead to leukemogenesis. In addition, it potentially provides a quite nice explanation for the interesting phenotype switch seen in this case.

Therapeutic Implications Mixed lineage acute leukemias are difficult to treat and have poor overall survival Aim therapy at myeloid or T component? Potential for targeted therapy aimed at suppressing TLX3 to overcome the differentiation “block” TLX3 is a good target bc it’s not expressed in most adult tissues.

Patient Follow-Up Switched from T-ALL to AML therapy Did not achieve second remission Died 2 months following AML diagnosis

Acknowledgements CHOP Gerald Wertheim Michele Paessler Jackie Biegel Penn Dale Frank Mayo Clinic Rhett Ketterling